Method of producing erythrocytes without feeder cells

ABSTRACT

Provided herein are methods of producing erythrocytes from hematopoietic cells, particularly hematopoietic cells from placental perfusate in combination with hematopoietic cells from umbilical cord blood, wherein the method results in accelerated expansion and differentiation of the hematopoietic cells to more efficiently produce administrable erythrocytes. Further provided herein is a bioreactor in which hematopoietic cell expansion and differentiation takes place.

This application claims benefit of U.S. Provisional Application No.61/222,930, filed Jul. 2, 2009, the disclosure of which is herebyincorporated by reference in its entirety.

1. FIELD

Provided herein, generally, are methods of expanding hematopoietic cellpopulations, e.g., CD34⁴ cell populations, and methods of producingadministrable units of erythrocytes from such cell populations. Alsoprovided herein is a bioreactor that accomplishes such expansion anddifferentiation.

2. BACKGROUND

Each year in the United States approximately 13 million units of bloodare used for transfusion or to generate life-saving blood products suchas platelets. Voluntary blood donation is utilized by the Red Cross andother agencies to procure from about 500 mL to about 1000 mL whole bloodsamples. Self-screening of voluntary donation is relatively safe andeffective in the US and Western Europe where the incidences of HIV andother adventitious pathogens are relatively low. However, in countriesin which HIV and hepatitis are endemic, procurement of safe blood fortransfusion can be highly problematic. As an alternative to voluntaryblood donation many groups have attempted to develop safe artificialblood substitutes that could undergo long-term storage. While some ofthese products show significant promise in transiently treatingtraumatic blood loss, such products are not designed for long-termsubstitution of red blood cell function. Increasingly there is a need todevelop a safe and plentiful supply of erythrocytes that can beadministered to patients on the battlefield or civilian hospitalsettings around the world.

Conventional methods for producing erythrocytes are either inefficient,too small in scale, or too laborious to allow for the continuous,on-site production of erythrocytes. Conventional dish or flask-basedculture systems are associated with discontinuous medium exchange, andgenerally dish-based culture systems cannot be used to handle singlebatches of >10⁹ cells. A logical further development from dishes are bagtechnologies, e.g. the Wave Bioreactor, in which the medium volume issignificantly enlarged by using bags and cell attachment surface can beenlarged by using buoyant carriers. However, bag-type reactors typicallyoperate from 2×10⁶ to about 6×10⁶ cells/ml medium, requiring significantmedia dilution during culture and a laborious 10-100 fold debulking.Moreover, bag technologies, and generally all large-vessel stirred tanktype bioreactors, do not provide tissue-like physiologic environmentsthat are conducive to “normal” cell expansion and differentiation.

3. SUMMARY

Provided herein are methods of expanding hematopoietic cells (e.g.,hematopoietic stem cells or hematopoietic progenitor cells), todifferentiating the expanded hematopoietic cells into administrableerythrocytes (red blood cells), and to the production of administrableunits of cells comprising the erythrocytes.

In one aspect, provided herein is a method of producing erythrocytes. Inone embodiment, the method comprises differentiating hematopoietic cellsfrom human placental perfusate to erythrocytes, wherein the methodcomprises expanding a population of hematopoietic cells in the absenceof a feeder layer; and differentiating the hematopoietic cells toerythrocytes or progenitors of erythrocytes.

In another aspect, provided herein is a bioreactor for the expansion ofhematopoietic cells and differentiation of said hematopoietic cells intoerythrocytes. The bioreactor allows for production of a number oferythrocytes equivalent to current methods of producing erythrocytes, ina much smaller volume, by facilitating a continuous erythrocyteproduction method rather than a batch method. In specific embodiments,the bioreactor comprises a cell culture element, a cell separationelement, a gas provision element and/or a medium provision element. In aspecific embodiment of the bioreactor, the erythrocytes are collected bymagnetic bead separation. In another embodiment of the method, theerythrocytes are collected by partially or fully deoxygenatinghemoglobin in said erythrocytes, and attracting the erythrocytes to asurface using a magnetic field.

In another aspect, provided herein is a method of the production oferythrocytes using the bioreactor described herein. In a specificembodiment, provided herein is a method of producing erythrocytescomprising producing erythrocytes using a plurality of the bioreactorsdisclosed herein. In other specific embodiments of the method, theproduction of said erythrocytes is automated.

In one aspect, provided herein is a method of producing erythrocytes,comprising expanding a population of hematopoietic cells in a medium inthe absence of feeder cells, wherein a plurality of hematopoietic cellswithin said population of hematopoietic cells differentiate intoerythrocytes during said expanding; and isolating said erythrocytes fromsaid medium, wherein said medium comprises SCF at a concentration ofabout 10 to about 10,000 ng/mL, IL-3 at a concentration of about 0.01 toabout 500 ng/mL, and EPO at a concentration of about 0.1 to about 10IU/mL, and wherein said SCF, IL-3 and Epo are not comprised within anundefined component of said medium (e.g., serum). In a specificembodiment of the method, said medium does not comprise one or more, orany, of Flt-3L, IL-11, thrombopoietin (Tpo), homeobox-B4 (HoxB4), ormethylcellulose. In other specific embodiments, said medium comprisesSCF at a concentration of about 20 to about 2000 ng/mL; about 50 toabout 1000 ng/mL; or about 100 ng/mL. In other specific embodiments,said medium comprises IL-3 at a concentration of about 0.1 to about 100ng/mL; about 1 to about 50 ng/mL; or about 5 ng/mL. In other specificembodiments, said medium comprises EPO at a concentration of about 1 toabout 5 IU/mL; or about 2 to about 3 IU/mL.

In another specific embodiment of the method, said medium furthercomprises insulin-like growth factor 1 (IGF-1) at a concentration ofabout 1 to about 1000 ng/mL and lipids at a concentration of about 1 toabout 1000 μg/mL, wherein said lipids comprise a mixture of protein andcholesterol; and wherein said medium comprises hydrocortisone at aconcentration of about 0.01 to about 100 μM, or dexamethasone at aconcentration of about 0.01 μM to about 100 μM. In more specificembodiments, said medium comprises IGF-1 at a concentration of about 10to about 500 ng/mL; or about 20 to about 100 ng/mL. In other morespecific embodiments, said medium comprises lipids at a concentration ofabout 10 to about 500 ng/mL; or about 20 to about 100 ng/mL. In othermore specific embodiments, said medium comprises hydrocortisone at aconcentration of about 0.1 to about 50 μM; or about 0.5 to about 10 μM.In other more specific embodiments, said medium comprises dexamethasoneat a concentration of about 0.05 to about 20 μM; or about 0.1 to about10 μM.

In a more specific embodiment, the medium comprises about 100 ng/mL SCF,about 3 U/mL Epo, about 40 ng/mL IGF-1, about 5 ng/mL IL-3, about 1 μMDexamethasone, and 40 μg/ml lipids, wherein said lipids comprise amixture of protein and cholesterol. In another more specific embodiment,the medium comprises about 100 ng/mL SCF, about 2 U/mL Epo, about 40ng/mL IGF-1, about 5 ng/mL IL-3, about 1 μM hydrocortisone, and 50 ng/mllipids, wherein said lipids comprise a mixture of protein andcholesterol.

In certain other embodiments, hematopoietic cells, in certainembodiments, are expanded and differentiated, in continuous fashion, ina culture medium comprising SCF; Epo; IGF-1; lipids, wherein the lipidscomprise a mixture of proteins and cholesterol (e.g., Lipids CholesterolRich from adult bovine serum; Cat. No. C7305-1G, Sigma, St Louis, Mo.);and either hydrocortisone or dexamethasone. In specific embodiments,said medium comprises SCF at a concentration of about 10 to about 10,000ng/mL; about 20 to about 2000 ng/mL; about 50 to about 1000 ng/mL; about100 ng/mL; or about 100 ng/mL. In other specific embodiments, saidmedium comprises Epo at a concentration of about 1 to about 5 IU/mL; orabout 2 to about 3 IU/mL. In other specific embodiments, said mediumcomprises IGF-1 at a concentration of about 1 to about 1000 ng/mL; about10 to about 500 ng/mL; about 20 to about 100 ng/mL; or about 40 ng/mL.In other specific embodiments, said medium comprises said lipids at aconcentration of about 1 to about 1000 μg/mL; about 10 to about 500ng/mL; about 20 to about 100 ng/mL; or about 40 ng/mL. In other specificembodiments, said medium comprises hydrocortisone at a concentration ofabout 0.1 μM to about 10 μM; about 0.5 μM to about 5 μM; or about 1 μM.In other specific embodiments, said medium comprises dexamethasone at aconcentration of about 0.1 μM to about 10 μM; about 0.5 μM to about 5μM; or about 1 μM.

In another specific embodiment of the method, the medium comprises animmunomodulatory compound, wherein the immunomodulatory compoundincreases the number of hematopoietic cells compared to a plurality ofhematopoietic cells expanded in the absence of the immunomodulatorycompound.

In a specific embodiment of any of the above media, the medium isserum-free.

In another specific embodiment of the method, said hematopoietic cellsare CD34⁺. In another specific embodiment, said hematopoietic cells areThy-1⁺, CXCR4⁺, CD133⁺ or KDR⁺. In another specific embodiment, saidhematopoietic cells are CD34⁻CD133⁺ or CD34⁻CD117⁺. In another specificembodiment, said hematopoietic cells are CD45⁻. In another specificembodiment, hematopoietic cells are CD2⁻, CD3⁻, CD11b⁻, CD11c⁻, CD14⁻,CD16⁻, CD19⁺, CD24⁻, CD56⁻, CD66b⁻ and/or glycophorin A⁻.

In another specific embodiment, said hematopoietic cells are obtainedfrom cord blood, placental blood, peripheral blood, bone marrow,embryonic stem cells or induced pluripotent cells. In another specificembodiment, said hematopoietic cells are obtained from placentalperfusate. In another specific embodiment, said hematopoietic cells areobtained from umbilical cord blood and placental perfusate. In a morespecific embodiment, said placental perfusate is obtained by passage ofperfusion solution through only the vasculature of a placenta. Inanother specific embodiment, said hematopoietic cells are humanhematopoietic cells.

In certain embodiments of the method, a plurality of said hematopoieticcells is blood type A, blood type O, blood type AB, blood type O; is Rhpositive or Rh negative; blood type M, blood type N, blood type S, orblood type s; blood type P1; blood type Lua, blood type Lub, or bloodtype Lu(a); blood type K (Kell), k (cellano), Kpa, Kpb, K(a+), Kp(a−b−)or K−k−Kp(a−b−); blood type Le(a−b−), Le(a+b−) or Le(a−b+); blood typeFy a, Fy b or Fy(a−b−); or blood type Jk(a−b−), Jk(a+b−), Jk(a−b+) orJk(a+b+). In a more specific embodiment, the hematopoietic cells aretype O, Rh positive; type O, Rh negative; type A, Rh positive; type A,Rh negative; type B, Rh positive; type B, Rh negative; type AB, Rhpositive or type AB, Rh negative. In other specific embodiments of themethod, greater than 90%, 95%, 98%, or 99%, or each, of saidhematopoietic cells is blood type A, blood type O, blood type AB, bloodtype O; is Rh positive or Rh negative; blood type M, blood type N, bloodtype S, or blood type s; blood type P1; blood type Lua, blood type Lub,or blood type Lu(a); blood type K (Kell), k (cellano), Kpa, Kpb, K(a+),Kp(a−b−) or K-k-Kp(a−b−); blood type Le(a−b−), Le(a+b−) or Le(a−b+);blood type Fy a, Fy b or Fy(a−b−); or blood type Jk(a−b−), Jk(a+b−),Jk(a−b+) or Jk(a+b+). In more specific embodiments, the hematopoieticcells are type O, Rh+; type O, Rh negative; type A, Rh positive; type A,Rh negative; type B, Rh positive; type B, Rh negative; type AB, Rhpositive or type AB, Rh negative.

Further provided herein are compositions, e.g., compositions comprisingerythrocytes, made by any of the methods described above. In a specificembodiment of the compositions, the percentage of cells in saidcomposition having fetal hemoglobin relative to the total number ofcells having hemoglobin is about 70 to about 99%. In another specificembodiment, the percentage of cells having adult hemoglobin relative tothe total number of cells having hemoglobin is about 5 to about 40%.

In certain embodiments, isolation of erythrocytes from medium in whichthe erythrocytes differentiate is performed continuously. In otherspecific embodiments, isolation of erythrocytes is performedperiodically, e.g., every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 55 or 60 minutes, or every 1, 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0,11.5, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, ormore during expansion and differentiation of the hematopoietic cells. Inanother specific embodiment, said isolation of erythrocytes is performedperiodically when one or more culture condition criteria are met, e.g.,achievement in the culture of a particular cell density; achievement inthe culture of a particular number of cells per milliliter expressingcertain erythrocyte markers, e.g., CD36 or glycophorin A; or the like.

In another aspect, provided herein is medium for growth ordifferentiation of hematopoietic cells, wherein said medium comprisesstem cell factor SCF at a concentration of about 10 to about 10,000ng/mL, IL-3 at a concentration of about 0.01 to about 500 ng/mL, and EPOat a concentration of about 0.1 to about 10 IU/mL, and wherein said SCF,IL-3 and Epo are not comprised within an undefined component of saidmedium (e.g., serum). In a specific embodiment, said medium does notcomprise one or more, or any, of Flt-3L, IL-11, thrombopoietin (Tpo),homeobox-B4 (HoxB4), or methylcellulose. In other specific embodiments,said medium comprises SCF at a concentration of about 20 to about 2000ng/mL; about 50 to about 1000 ng/mL; or about 100 ng/mL. In otherspecific embodiments, said medium comprises IL-3 at a concentration ofabout 0.1 to about 100 ng/mL; about 1 to about 50 ng/mL; or about 5ng/mL. In other specific embodiments, said medium comprises EPO at aconcentration of about 1 to about 5 IU/mL; or about 2 to about 3 IU/mL.

In another specific embodiment, said medium further comprisesinsulin-like growth factor 1 (IGF-1) at a concentration of about 1 toabout 1000 ng/mL and lipids at a concentration of about 1 to about 1000μg/mL, wherein said lipids comprise a mixture of protein andcholesterol; and wherein said medium comprises hydrocortisone at aconcentration of about 0.01 to about 100 μM, or dexamethasone at aconcentration of about 0.01 μM to about 100 μM. In more specificembodiments, said medium comprises IGF-1 at a concentration of about 10to about 500 ng/mL; or about 20 to about 100 ng/mL. In other morespecific embodiments, said medium comprises lipids at a concentration ofabout 10 to about 500 ng/mL; or about 20 to about 100 ng/mL. In othermore specific embodiments, said medium comprises hydrocortisone at aconcentration of about 0.1 to about 50 μM; or about 0.5 to about 10 μM.In other more specific embodiments, said medium comprises dexamethasoneat a concentration of about 0.05 to about 20 μM; or about 0.1 to about10 μM.

In a more specific embodiment, the medium comprises about 100 ng/mL SCF,about 3 U/mL Epo, about 40 ng/mL IGF-1, about 5 ng/mL IL-3, about 1 μMDexamethasone, and 40 μg/mL lipids, wherein said lipids comprise amixture of protein and cholesterol. In another more specific embodiment,the medium comprises about 100 ng/mL SCF, about 2 U/mL Epo, about 40ng/mL IGF-1, about 5 ng/mL IL-3, about 1 μM hydrocortisone, and 50 ng/mllipids, wherein said lipids comprise a mixture of protein andcholesterol.

In another specific embodiment, the medium comprises Iscove's ModifiedDulbecco's Medium or RPMI and is further supplemented with 1% BovineSerum Albumin; 10 microgram/mL Recombinant Human Insulin; 100microgram/mL Human Transferrin (Iron saturated); and 0.1 μM2-Mercaptoethanol; 2 mM L-glutamine.

In certain other embodiments, the medium comprises SCF; Epo; IGF-1;lipids, wherein the lipids comprise a mixture of proteins andcholesterol (e.g., Lipids Cholesterol Rich from adult bovine serum; Cat.No. C7305-1G, Sigma, St Louis, Mo.); and either hydrocortisone ordexamethasone. In specific embodiments, said medium comprises SCF at aconcentration of about 10 to about 10,000 ng/mL; about 20 to about 2000ng/mL; about 50 to about 1000 ng/mL; about 100 ng/mL; or about 100ng/mL. In other specific embodiments, said medium comprises Epo at aconcentration of about 1 to about 5 IU/mL; or about 2 to about 3 IU/mL.In other specific embodiments, said medium comprises IGF-1 at aconcentration of about 1 to about 1000 ng/mL; about 10 to about 500ng/mL; about 20 to about 100 ng/mL; or about 40 ng/mL. In other specificembodiments, said medium comprises said lipids at a concentration ofabout 1 to about 1000 μg/mL; about 10 to about 500 ng/mL; about 20 toabout 100 ng/mL; or about 40 ng/mL. In other specific embodiments, saidmedium comprises hydrocortisone at a concentration of about 0.1 μM toabout 10 μM; about 0.5 μM to about 5 μM; or about 1 μM. In otherspecific embodiments, said medium comprises dexamethasone at aconcentration of about 0.1 μM to about 10 μM; about 0.5 μM to about 5μM; or about 1 μM.

As used herein, the term “hematopoietic cells” includes hematopoieticstem cells and hematopoietic progenitor cells, that is, blood cells ableto differentiate into erythrocytes.

As used herein, “+”, when used to indicate the presence of a particularcellular marker, means that the cellular marker is detectably present influorescence activated cell sorting over an isotype control; or isdetectable above background in quantitative or semi-quantitative RT-PCR.

As used herein, “−”, when used to indicate the presence of a particularcellular marker, means that the cellular marker is not detectablypresent in fluorescence activated cell sorting over an isotype control;or is not detectable above background in quantitative orsemi-quantitative RT-PCR.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Flow cytometric analysis of HPP-derived CD34⁺/CD45⁻ andCD34⁺/CD45⁺ cells.

FIGS. 2A, 2B: Flow cytometric analysis of recovered CD34⁺ cells. (A):CD34⁺ cells prior to isolation; (B) CD34⁺ cells after isolation.

FIGS. 3A, 3B: Cell expansion in pomalidomide supplemented IMDM medium.FIG. 3A: Fold expansion of total nucleated cells (TNC). FIG. 3B: Foldexpansion of CD34⁺ cells.

FIGS. 4A-4C: Expandability of CD34⁺ cultures. FIG. 4A: Fold expansion ofTNC. FIG. 4B: Fold expansion of CD34⁺ cells. FIG. 4C: Expansion of CD34⁺cells in number of cells. “Compound 1” is pomalidomide.

FIG. 5: Cell expansion in medium formulations C and E1.

FIG. 6: Medium optimization—E1, E2, E3 and E4. Error bars represent thestandard deviation calculated for population means for 3 donors.

FIG. 7: Effects of cell density on cell expansion.

FIG. 8: Comparison of proliferation potential of BM and CB derived CD34+cells. Standard deviation was calculated for population means for 3donors.

FIGS. 9A, 9B: Long term cultures in E3 medium. (A) Cell fold expansion;(B) Population doublings.

FIGS. 10A, 10B: ELISA analysis of HbF and HbA production. (A) HbFproduction; (B) HbA production. Standard deviation was calculated formeans for 3 replicates.

FIG. 11: 3-level design of experiment (DOE) study to delineateinteractions among SCF, Epo and IL-3. Full factorial experiment design.

FIGS. 12A-12C: 3-level DOE study to delineate interactions among SCF,Epo and IL-3. Cube plot for factorial effects on cell differentiation(FIG. 12A); interaction plots of SCF (FIG. 12B) and Epo (FIG. 12C) oncell expansion and differentiation.

FIG. 13A-13C: 3-level DOE study to delineate interactions among SCF, Epoand cell density. FIG. 13A: Cube plot for factorial effects on cellexpansion and differentiation; Interaction plots of SCF and cell densityon cell expansion and differentiation at low cell density, high SCFconcentration (FIG. 13B) or high cell density, low SCF concentration(FIG. 13C).

FIG. 14: Erythrocyte sorting by cell size. Erythrocyte population (P1)can be distinguished from other immature populations (P2 and P3) usingforward (FSC) and side scatter (SSC).

FIG. 15: Erythrocyte sorting by DRAQ staining.

5. DETAILED DESCRIPTION

Provided herein is a method of producing erythrocytes from expandedhematopoietic cells, e.g., hematopoietic stem cells and/or hematopoieticprogenitor cells. In one embodiment, hematopoietic cells are collectedfrom a source of such cells, e.g., placental perfusate, umbilical cordblood, placental blood, peripheral blood, and/or bone marrow. Thehematopoietic cells are expanded and differentiated, continuously,without the use of feeder cells. Such isolation, expansion anddifferentiation can be performed in a central facility, which providesexpanded hematopoietic cells for shipment to decentralized expansion anddifferentiation at points of use, e.g., hospital, military base,military front line, or the like. Collection of erythrocytes produced inthe method, in a preferred embodiment, is performed continuously orperiodically, e.g., during differentiation. The continuous or periodicseparation aspect of the method allows for the production oferythrocytes in a substantially smaller space than possible using, e.g.,batch methods. The time for collection and expansion of thehematopoietic cells is approximately 5-10 days, typically about 7 days.The time for expansion and differentiation of the hematopoietic cellsinto erythrocytes is approximately 21-28 days. Erythrocytes, in certainembodiments, are then purified on-site and packaged into administrableunits.

In one aspect, provided herein is a method of producing erythrocytes,comprising expanding a population of hematopoietic cells in a medium inthe absence of feeder cells, wherein a plurality of hematopoietic cellswithin said population of hematopoietic cells differentiate intoerythrocytes during said expanding; and isolating said erythrocytes fromsaid medium, wherein said medium comprises SCF at a concentration ofabout 10 to about 10,000 ng/mL, IL-3 at a concentration of about 0.01 toabout 500 ng/mL, and EPO at a concentration of about 0.1 to about 10IU/mL, and wherein said SCF, IL-3 and Epo are not comprised within anundefined component of said medium (e.g., serum). In a specificembodiment, said isolating of erythrocytes in step (c) is performedcontinuously. In other specific embodiments, said isolating oferythrocytes in step (c) is performed periodically, e.g., every 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes,or every 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0,8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23 or 24 hours, or more. In another specific embodiment,said isolating of erythrocytes in step (c) is performed periodicallywhen one or more culture condition criteria are met, e.g., achievementin the culture of a particular cell density; achievement in the cultureof a particular number of cells per milliliter expressing certainerythrocyte markers, e.g., CD36 or glycophorin A; or the like. Themethod of expanding and differentiating the hematopoietic cells isdescribed in more detail in Section 5.2.2, below.

5.1. Hematopoietic Cells

Hematopoietic cells useful in the methods disclosed herein can be anyhematopoietic cells able to differentiate into erythrocytes, e.g.,precursor cells, hematopoietic progenitor cells, hematopoietic stemcells, or the like. Hematopoietic cells can be obtained from tissuesources such as, e.g., bone marrow, cord blood, placental blood,peripheral blood, or the like, or combinations thereof. Hematopoieticcells can be obtained from placenta. In a specific embodiment, thehematopoietic cells are obtained from placental perfusate. Hematopoieticcells from placental perfusate can comprise a mixture of fetal andmaternal hematopoietic cells, e.g., a mixture in which maternal cellscomprise greater than 5% of the total number of hematopoietic cells.Preferably, hematopoietic cells from placental perfusate comprise atleast about 90%, 95%, 98%, 99% or 99.5% fetal cells.

In certain embodiments, the hematopoietic cells are CD34⁺ cells. CD34⁺hematopoietic cells can, in certain embodiments, express or lack thecellular marker CD38. Thus, in specific embodiments, the hematopoieticcells useful in the methods disclosed herein are CD34⁺CD38⁺ orCD34⁺CD38⁻. In a more specific embodiment, the hematopoietic cells areCD34⁺CD38⁻Lin⁻. In another specific embodiment, the hematopoietic cellis one or more of CD2⁻, CD3⁻, CD11b⁻, CD11c⁻, CD14⁻, CD16⁻, CD19⁻,CD24⁻, CD56⁻, CD66b⁻ and glycophorin A⁻. In another specific embodiment,the hematopoietic cell is CD2⁻, CD3⁻, CD11b⁻, CD11c⁻, CD14⁻, CD16⁻,CD19⁻, CD24⁻, CD56⁻, CD66b⁻ and glycophorin A⁻. In other specificembodiments, the hematopoietic cells are CD34⁺ and CD133⁺; CD34⁺ andCD133⁻; CD34⁺ and CD117⁺; or CD34⁺ and CD117⁻. In another more specificembodiment, the hematopoietic cell is CD34⁺CD38⁻CD33⁻CD117⁻. In anothermore specific embodiment, the hematopoietic cell isCD34⁺CD38⁻CD33⁻CD117⁻CD235⁻CD36⁻.

In another embodiment, the hematopoietic cells are CD45⁻. In a specificembodiment, the hematopoietic cells are CD34⁺CD45⁻. In another specificembodiment, the hematopoietic cells are CD34⁺CD45⁺.

In another embodiment, the hematopoietic cell is Thy-1⁺. In a specificembodiment, the hematopoietic cell is CD34⁺Thy-1⁺. In anotherembodiment, the hematopoietic cells are CD133⁺. In specific embodiments,the hematopoietic cells are CD34⁺CD133⁺ or CD133⁺Thy-1⁺. In anotherspecific embodiment, the CD34⁺ hematopoietic cells are CXCR4⁺. Inanother specific embodiment, the CD34⁺ hematopoietic cells are CXCR4⁻.In another embodiment, the hematopoietic cells are positive for KDR(vascular growth factor receptor 2). In specific embodiments, thehematopoietic cells are CD34⁺KDR⁺, CD133⁺KDR⁺ or Thy-1⁺KDR⁺. In certainother embodiments, the hematopoietic cells are positive for aldehydedehydrogenase (ALDH⁺), e.g., the cells are CD34⁺ALDH⁺.

In certain embodiments, the hematopoietic cells are CD34⁻.

The hematopoietic cells can also lack certain markers that indicatelineage commitment, or a lack of developmental naiveté. For example, inanother embodiment, the hematopoietic cells are HLA-DR⁻. In specificembodiments, the hematopoietic cells are CD34⁺HLA-DR⁻, CD133⁺HLA-DR⁻,Thy-1⁺HLA-DR⁻ or ALDH⁺HLA-DR⁻ In another embodiment, the hematopoieticcells are negative for one or more, preferably all, of lineage markersCD2, CD3, CD11b, CD11c, CD14, CD16, CD19, CD24, CD56, CD66b andglycophorin A.

Thus, populations of hematopoietic cells can be selected for use in themethods disclosed herein on the basis of the presence of markers thatindicate an undifferentiated state, or on the basis of the absence oflineage markers indicating that at least some lineage differentiationhas taken place. Methods of isolating cells on the basis of the presenceor absence of specific markers is discussed in detail, e.g., in Section5.1.2, below.

Hematopoietic cells used in the methods provided herein can be asubstantially homogeneous population, e.g., a population comprising atleast about 95%, at least about 98% or at least about 99% hematopoieticcells from a single tissue source, or a population comprisinghematopoietic cells exhibiting the same hematopoietic cell-associatedcellular markers. For example, in various embodiment, the hematopoieticcells can comprise at least about 95%, 98% or 99% hematopoietic cellsfrom bone marrow, cord blood, placental blood, peripheral blood, orplacenta, e.g., placenta perfusate.

Hematopoietic cells used in the methods provided herein can be obtainedfrom a single individual, e.g., from a single placenta, or from aplurality of individuals, e.g., can be pooled. Where the hematopoieticcells are obtained from a plurality of individuals and pooled, it ispreferred that the hematopoietic cells be obtained from the same tissuesource. Thus, in various embodiments, the pooled hematopoietic cells areall from placenta, e.g., placental perfusate, all from placental blood,all from umbilical cord blood, all from peripheral blood, and the like.

Hematopoietic cells used in the methods disclosed herein can comprisehematopoietic cells from two or more tissue sources. Preferably, whenhematopoietic cells from two or more sources are combined for use in themethods herein, a plurality of the hematopoietic cells used to produceerythrocytes comprise hematopoietic cells from placenta, e.g., placentaperfusate. In various embodiments, the hematopoietic cells used toproduce erythrocytes comprise hematopoietic cells from placenta and fromcord blood; from placenta and peripheral blood; form placenta andplacental blood, or placenta and bone marrow. In a preferred embodiment,the hematopoietic cells comprise hematopoietic cells from placentalperfusate in combination with hematopoietic cells from cord blood,wherein the cord blood and placenta are from the same individual, i.e.,wherein the perfusate and cord blood are matched. In embodiments inwhich the hematopoietic cells comprise hematopoietic cells from twotissue sources, the hematopoietic cells from the sources can be combinedin a ratio of, for example, 1:10, 2:9, 3:8, 4:7, 5:6, 6:5, 7:4, 8:3,9:2, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1 or 9:1.

Preferably, the erythrocytes produced from hematopoietic cells accordingto the methods provided herein are homogeneous with respect to bloodtype, e.g., identical with respect to cell surface markers, antigens, orthe like. Such homogeneity can be achieved, for example, by obtaininghematopoietic cells from a single individual of the desired blood type.In embodiments in which hematopoietic cells are pooled from a pluralityof individuals, it is preferred that each of the individuals shares atleast one, at least two, or at least three or more antigenic blooddeterminants in common. In various embodiments, for example, theindividual from which the hematopoietic cells are obtained is, or eachof the individuals from which hematopoietic cells are obtained are,blood type O, blood type A, blood type B, or blood type AB. In otherembodiments, the individual from which the hematopoietic cells areobtained is, or each of the individuals from which hematopoietic cellsare obtained are, Rh positive, or Rh negative. In a specific embodiment,the individual from which the hematopoietic cells are obtained is, oreach of the individuals from which hematopoietic cells are obtained are,O positive and Rh negative. In more specific embodiments, the individualfrom which the hematopoietic cells are obtained is, or each of theindividuals from which hematopoietic cells are obtained are, O positive,O negative, A positive, A negative, B positive, B negative, AB positive,or AB negative. In other specific embodiments, the individual from whichthe hematopoietic cells are obtained is, or each of the individuals fromwhich hematopoietic cells are obtained are, blood type M, blood type N,blood type S, or blood type s. In other specific embodiments, theindividual from which the hematopoietic cells are obtained is, or eachof the individuals from which hematopoietic cells are obtained are,blood type P1. In other specific embodiments, the individual from whichthe hematopoietic cells are obtained is, or each of the individuals fromwhich hematopoietic cells are obtained are, blood type Lua, blood typeLub, or blood type Lu(a). In other specific embodiments, the individualfrom which the hematopoietic cells are obtained is, or each of theindividuals from which hematopoietic cells are obtained are, blood typeK (Kell), k (cellano), Kpa, Kpb, K(a+), Kp(a−b−) or K-k-Kp(a−b−). Inother specific embodiments, the individual from which the hematopoieticcells are obtained is, or each of the individuals from whichhematopoietic cells are obtained are, blood type Le(a−b−), Le(a+b−) orLe(a−b+). In other specific embodiments, the individual from which thehematopoietic cells are obtained is, or each of the individuals fromwhich hematopoietic cells are obtained are, blood type Fy a, Fy b orFy(a−b−). In other specific embodiments, the individual from which thehematopoietic cells are obtained is, or each of the individuals fromwhich hematopoietic cells are obtained are, blood type Jk(a−b−),Jk(a+b−), Jk(a−b+) or Jk(a+b+). In other specific embodiments, theindividual from whom the hematopoietic cells are obtained isclassifiable within blood group Diego, Cartwright, Xgm Scianna,Bombrock, Colton, Lansteiner-Weiner, Chido/Rogers, Hh, Kx, Gergich,Cromer, Knops, Indian, Ok, Raph, or JMH. In other specific embodiments,each of the individuals from which hematopoietic cells are obtained areof the same blood type within a blood typing system or group ofantigenic determinants, wherein said blood typing system or group ofantigenic determinants are Diego, Cartwright, Xgm Scianna, Bombrock,Colton, Lansteiner-Weiner, Chido/Rogers, Hh, Kx, Gergich, Cromer, Knops,Indian, Ok, Raph, or JMH.

5.1.1. Placental Hematopoietic Stem Cells

In certain embodiments, the hematopoietic cells used in the methodsprovided herein are placental hematopoietic cells. As used herein,“placental hematopoietic cells” means hematopoietic cells obtained fromthe placenta itself, and not from placental blood or from umbilical cordblood. In one embodiment, placental hematopoietic cells are CD34⁺. In aspecific embodiment, the placental hematopoietic cells are predominantly(e.g., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or 98%) CD34⁺CD38⁻ cells. In another specific embodiment, the placentalhematopoietic cells are predominantly (e.g., at least about 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%) CD34⁺CD38⁺ cells.Placental hematopoietic cells can be obtained from a post-partummammalian (e.g., human) placenta by any means known to those of skill inthe art, e.g., by perfusion.

In another embodiment, the placental hematopoietic cell is CD45⁻. In aspecific embodiment, the hematopoietic cell is CD34⁺CD45⁻. In anotherspecific embodiment, the placental hematopoietic cells are CD34⁺CD45⁺.

5.1.1.1. Obtaining Placental Hematopoietic Cells by Perfusion

Placental hematopoietic cells can be obtained using perfusion. Methodsof perfusing mammalian placenta to obtain cells, including placentalhematopoietic cells, are disclosed, e.g., in U.S. Pat. No. 7,045,148,entitled “Method of Collecting placental Stem Cells,” U.S. Pat. No.7,255,879, entitled “Post-Partum Mammalian Placenta, Its Use andPlacental Stem Cells Therefrom,” and in U.S. Application No.2007/0190042, entitled “Improved Medium for Collecting Placental StemCells and Preserving Organs,” the disclosures of which are herebyincorporated by reference in their entireties.

Placental hematopoietic cells can be collected by perfusion, e.g.,through the placental vasculature, using, e.g., a saline solution (forexample, phosphate-buffered saline, a 0.9% NaCl solution, or the like),culture medium or organ preservation solution as a perfusion solution.In one embodiment, a mammalian placenta is perfused by passage ofperfusion solution through either or both of the umbilical artery andumbilical vein. The flow of perfusion solution through the placenta maybe accomplished using, e.g., gravity flow into the placenta. Preferably,the perfusion solution is forced through the placenta using a pump,e.g., a peristaltic pump. The umbilical vein can be, e.g., cannulatedwith a cannula, e.g., a TEFLON® or plastic cannula, which is connectedto a sterile connection apparatus, such as sterile tubing, which, inturn is connected to a perfusion manifold.

In preparation for perfusion, the placenta is preferably oriented (e.g.,suspended) in such a manner that the umbilical artery and umbilical veinare located at the highest point of the placenta. The placenta can beperfused by passage of a perfusion fluid through the placentalvasculature and surrounding tissue. The placenta can also be perfused bypassage of a perfusion fluid into the umbilical vein and collection fromthe umbilical arteries, or passage of a perfusion fluid into theumbilical arteries and collection from the umbilical vein.

In one embodiment, for example, the umbilical artery and the umbilicalvein are connected simultaneously, e.g., to a pipette that is connectedvia a flexible connector to a reservoir of the perfusion solution. Theperfusion solution is passed into the umbilical vein and artery. Theperfusion solution exudes from and/or passes through the walls of theblood vessels into the surrounding tissues of the placenta, and iscollected in a suitable open vessel, e.g., a sterile pan, from thesurface of the placenta that was attached to the uterus of the motherduring gestation. The perfusion solution may also be introduced throughthe umbilical cord opening and allowed to flow or percolate out ofopenings in the wall of the placenta which interfaced with the maternaluterine wall. Placental cells that are collected by this method, whichcan be referred to as a “pan” method, are typically a mixture of fetaland maternal cells.

In another embodiment, the perfusion solution is passed through theumbilical veins and collected from the umbilical artery, or is passedthrough the umbilical artery and collected from the umbilical veins.Placental cells collected by this method, which can be referred to as a“closed circuit” method, are typically almost exclusively fetal.

The closed circuit perfusion method can, in one embodiment, be performedas follows. A post-partum placenta is obtained within about 48 hoursafter birth. The umbilical cord is clamped and cut above the clamp. Theumbilical cord can be discarded, or can processed to recover, e.g.,umbilical cord stem cells, and/or to process the umbilical cord membranefor the production of a biomaterial. The amniotic membrane can beretained during perfusion, or can be separated from the chorion, e.g.,using blunt dissection with the fingers. If the amniotic membrane isseparated from the chorion prior to perfusion, it can be, e.g.,discarded, or processed, e.g., to obtain stem cells by enzymaticdigestion, or to produce, for example, an amniotic membrane biomaterial,e.g., the biomaterial described in U.S. Application Publication No.2004/0048796, the disclosure of which is hereby incorporated byreference in its entirety.

After cleaning the placenta of all visible blood clots and residualblood, e.g., using sterile gauze, the umbilical cord vessels areexposed, e.g., by partially cutting the umbilical cord membrane toexpose a cross-section of the cord. The vessels are identified, andopened, e.g., by advancing a closed alligator clamp through the cut endof each vessel. The apparatus, e.g., plastic tubing connected to aperfusion device or peristaltic pump, is then inserted into each of theplacental arteries. The pump can be any pump suitable for the purpose,e.g., a peristaltic pump. Plastic tubing, connected to a sterilecollection reservoir, e.g., a blood bag such as a 250 mL collection bag,is then inserted into the placental vein. Alternatively, the tubingconnected to the pump is inserted into the placental vein, and tubes toa collection reservoir(s) are inserted into one or both of the placentalarteries. The placenta is then perfused with a volume of perfusionsolution, e.g., about 750 ml of perfusion solution. Cells in theperfusate are then collected, e.g., by centrifugation.

In one embodiment, the proximal umbilical cord is clamped duringperfusion, and more preferably, is clamped within 4-5 cm (centimeter) ofthe cord's insertion into the placental disc.

The first collection of perfusion fluid from a mammalian placenta duringthe exsanguination process is generally colored with residual red bloodcells of the cord blood and/or placental blood. The perfusion fluidbecomes more colorless as perfusion proceeds and the residual cord bloodcells are washed out of the placenta. Generally from 30 to 100 ml(milliliter) of perfusion fluid is adequate to initially exsanguinatethe placenta, but more or less perfusion fluid may be used depending onthe observed results.

The volume of perfusion liquid used to collect placental hematopoieticcells may vary depending upon the number of hematopoietic cells to becollected, the size of the placenta, the number of collections to bemade from a single placenta, etc. In various embodiments, the volume ofperfusion liquid may be from 50 mL to 5000 mL, 50 mL to 4000 mL, 50 mLto 3000 mL, 100 mL to 2000 mL, 250 mL to 2000 mL, 500 mL to 2000 mL, or750 mL to 2000 mL. Typically, the placenta is perfused with 700-800 mLof perfusion liquid following exsanguination.

The placenta can be perfused a plurality of times over the course ofseveral hours or several days to obtain placental hematopoietic cells.Where the placenta is to be perfused a plurality of times, it may bemaintained or cultured under aseptic conditions in a container or othersuitable vessel, and perfused with a stem cell collection composition(see U.S. Application Publication No. 2007/0190042, the disclosure ofwhich is incorporated herein by reference in its entirety), or astandard perfusion solution (e.g., a normal saline solution such asphosphate buffered saline (“PBS”)) with or without an anticoagulant(e.g., heparin, warfarin sodium, coumarin, bishydroxycoumarin), and/orwith or without an antimicrobial agent (e.g., β-mercaptoethanol (0.1mM); antibiotics such as streptomycin (e.g., at 40-100 μg/ml),penicillin (e.g., at 40 U/ml), amphotericin B (e.g., at 0.5 μg/ml). Inone embodiment, an isolated placenta is maintained or cultured for aperiod of time without collecting the perfusate, such that the placentais maintained or cultured for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours, or 2 or 3 or moredays before perfusion and collection of perfusate. The perfused placentacan be maintained for one or more additional time(s), e.g., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24 or more hours, and perfused a second time with, e.g., 700-800 mLperfusion fluid. The placenta can be perfused 1, 2, 3, 4, 5 or moretimes, for example, once every 1, 2, 3, 4, 5 or 6 hours. In a preferredembodiment, perfusion of the placenta and collection of perfusionsolution, e.g., stem cell collection composition, is repeated until thenumber of recovered nucleated cells falls below 100 cells/ml. Theperfusates at different time points can be further processedindividually to recover time-dependent populations of cells, e.g.,placental hematopoietic cells. Perfusates from different time points canalso be pooled.

5.1.1.2. Obtaining Placental Hematopoietic Cells by Tissue Disruption

Hematopoietic cells can be isolated from placenta by perfusion with asolution comprising one or more proteases or other tissue-disruptiveenzymes (e.g., trypsin, collagenase, papain, chymotrypsin, subtilisin,hyaluronidase; a cathepsin, a caspase, a calpain, chymosin, plasmepsin,pepsin, or the like). In a specific embodiment, a placenta or portionthereof (e.g., amniotic membrane, amnion and chorion, placental lobuleor cotyledon, umbilical cord, or combination of any of the foregoing) isbrought to 25° C.-37° C., and is incubated with one or moretissue-disruptive enzymes in 200 mL of a culture medium for 30 minutes.Cells from the perfusate are collected, brought to 4° C., and washedwith a cold inhibitor mix comprising 5 mM EDTA, 2 mM dithiothreitol and2 mM beta-mercaptoethanol. The stem cells are washed after severalminutes with cold (e.g., 4° C.) stem cell collection composition.

In one embodiment, the placenta can be disrupted mechanically (e.g., bycrushing, blending, dicing, mincing or the like) to obtain thehematopoietic cells. The placenta can be used whole, or can be dissectedinto components prior to physical disruption and/or enzymatic digestionand hematopoietic cell recovery. For example, hematopoietic cells can beobtained from the amniotic membrane, chorion, umbilical cord, placentalcotyledons, or any combination thereof.

Placental hematopoietic cells can also be obtained by enzymaticdisruption of the placenta using a tissue-disrupting enzyme, e.g.,trypsin, collagenase, papain, chymotrypsin, subtilisin, hyaluronidase; acathepsin, a caspase, a calpain, chymosin, plasmepsin, pepsin, or thelike. Enzymatic digestion preferably uses a combination of enzymes,e.g., a combination of a matrix metalloprotease and a neutral protease,for example, a combination of collagenase and dispase. In oneembodiment, enzymatic digestion of placental tissue uses a combinationof a matrix metalloprotease, a neutral protease, and a mucolytic enzymefor digestion of hyaluronic acid, such as a combination of collagenase,dispase, and hyaluronidase or a combination of LIBERASE (BoehringerMannheim Corp., Indianapolis, Ind.) and hyaluronidase. Other enzymesthat can be used to disrupt placenta tissue include papain,deoxyribonucleases, serine proteases, such as trypsin, chymotrypsin, orelastase. Serine proteases may be inhibited by alpha 2 microglobulin inserum and therefore the medium used for digestion is usually serum-free.EDTA and DNase are commonly used in enzyme digestion procedures toincrease the efficiency of cell recovery. The digestate is preferablydiluted so as to avoid trapping stem cells within the viscous digest.

Any combination of tissue digestion enzymes can be used. Typicalconcentrations for tissue digestion enzymes include, e.g., 50-200 U/mLfor collagenase I and collagenase IV, 1-10 U/mL for dispase, and 10-100U/mL for elastase. Proteases can be used in combination, that is, two ormore proteases in the same digestion reaction, or can be usedsequentially in order to liberate placental stem cells. For example, inone embodiment, a placenta, or part thereof, is digested first with anappropriate amount of collagenase I at 2 mg/ml for 30 minutes, followedby digestion with trypsin, 0.25%, for 10 minutes, at 37° C. Serineproteases are preferably used consecutively following use of otherenzymes.

In another embodiment, the tissue can further be disrupted by theaddition of a chelator, e.g., ethylene glycol bis(2-aminoethylether)-N,N,N′N′-tetraacetic acid (EGTA) or ethylenediaminetetraaceticacid (EDTA) to the stem cell collection composition comprising the stemcells, or to a solution in which the tissue is disrupted and/or digestedprior to isolation of the placental hematopoietic cells.

It will be appreciated that where an entire placenta, or portion of aplacenta comprising both fetal and maternal cells (for example, wherethe portion of the placenta comprises the chorion or cotyledons), theplacental hematopoietic cells collected will comprise a mix of placentalstem cells derived from both fetal and maternal sources. Where a portionof the placenta that comprises no, or a negligible number of, maternalcells (for example, amnion), the placental stem cells collected willcomprise almost exclusively fetal placental stem cells.

5.1.2. Isolation, Sorting, and Characterization of Cells

Cells, including hematopoietic cells from any source, e.g., mammalianplacenta, can initially be purified from (i.e., be isolated from) othercells by, e.g., Ficoll gradient centrifugation, hetastarch treatment orammonium chloride treatment. Centrifugation, e.g., Ficoll (e.g., from GEHealthcare, Cat. No. 17-1440-03) centrifugation, can follow any standardprotocol for centrifugation speed, etc. In one embodiment, for example,cells collected from the placenta are recovered from perfusate bycentrifugation at 150×g for 15 minutes at room temperature, whichseparates cells from, e.g., contaminating debris and platelets. Inanother embodiment, placental perfusate is concentrated to about 200 ml,gently layered over Ficoll, and centrifuged at about 1100×g for 20minutes at about 22° C., and the low-density interface layer of cells iscollected for further processing.

In a specific, non-limiting embodiment, hetastarch (e.g., HetaSep, StemCell Technologies, Catalog No. 07906) treatment can be performed byadding 1 part hetastarch solution to 5 parts, e.g., human placentalperfusate (HPP) or cord blood in an appropriately sized tube. Aftermixing well, samples are allowed to settle until the plasma/RBCinterface is at approximately 50% of the total volume. Optionally,placing the tube in a 37° C. incubator for this step increases thesedimentation rate. A defined interface forms between the RBC fractionand the RBC-depleted (nucleated cell-rich) fraction as the RBC sedimentthrough the hetastarch solution. The leukocyte-rich layer is thenharvested and placed in a 50 mL tube. This fraction is washed once with,e.g., at least a four-fold volume of appropriate medium. A slow spin isperformed to remove platelets by centrifuging at, e.g., 120×g for 10minutes at room temperature (15-25° C.) with no brake. In certain otherembodiments, ammonium chloride (e.g., Stem Cell Technologies, CatalogNo. 07850) treatment can be performed by adding buffered ammoniumchloride solution (NH₄Cl) to HPP or cord blood e.g. at a volume:volumeratio of about 4:1. Vortex the cell suspension and place on ice for 10minutes to allow erythrocytes to lyse. Cells are optionally washed twicein the appropriate medium prior to use.

Cell pellets can be resuspended in, e.g., fresh saline solution, or amedium suitable for stem cell maintenance, e.g., IMDM serum-free mediumcontaining 2 U/ml heparin and 2 mM EDTA (GibcoBRL, NY). The totalmononuclear cell fraction can be isolated, e.g., using LYMPHOPREP®(Nycomed Pharma, Oslo, Norway) according to the manufacturer'srecommended procedure.

As used herein, “isolating” cells, including placental cells, e.g.,placental hematopoietic cells or placental stem cells, means to removeat least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of thecells with which the isolated cells are normally associated in theintact tissue, e.g., mammalian placenta. A cell from an organ is“isolated” when the cell is present in a population of cells thatcomprises fewer than 50% of the cells with which the stem cell isnormally associated in the intact organ.

The number and type of cells collected from a mammalian placenta can bemonitored, for example, by measuring changes in morphology and cellsurface markers using standard cell detection techniques such as flowcytometry, cell sorting, immunocytochemistry (e.g., staining with tissuespecific or cell-marker specific antibodies) fluorescence activated cellsorting (FACS), magnetic activated cell sorting (MACS), by examinationof the morphology of cells using light or confocal microscopy, and/or bymeasuring changes in gene expression using techniques well known in theart, such as PCR and gene expression profiling. These techniques can beused, too, to identify cells that are positive for one or moreparticular markers. For example, using antibodies to CD34, one candetermine, using the techniques above, whether a cell comprises adetectable amount of CD34, in an assay such as an ELISA or RIA, or byFACS; if so, the cell is CD34⁺. Similarly, if a cell, produces enoughRNA encoding, e.g., OCT-4 to be detectable by RT-PCR, the cell isOCT-4⁺. Antibodies to cell surface markers (e.g., CD markers such asCD34) and the sequence of stem cell-specific genes, such as OCT-4, arewell-known in the art.

Placental cells, particularly cells that have been isolated, e.g., byFicoll separation, hetastarch treatment, ammonium chloride treatment,differential adherence, or a combination of both, may be sorted usingfluorescence activated cell sorting (FACS). FACS is a well-known methodfor separating particles, including cells, based on the fluorescentproperties of the particles (Kamarch, 1987, Methods Enzymol,151:150-165). Laser excitation of fluorescent moieties in the individualparticles results in a small electrical charge allowing electromagneticseparation of positive and negative particles from a mixture. In oneembodiment, cell surface marker-specific antibodies or ligands arelabeled with distinct fluorescent labels. Cells are processed throughthe cell sorter, allowing separation of cells based on their ability tobind to the antibodies used. FACS sorted particles may be directlydeposited into individual wells of 96-well or 384-well plates tofacilitate separation and cloning.

In one embodiment, stem cells from placenta are sorted, e.g., isolated,on the basis of expression one or more of the markers CD34, CD38, CD44,CD45, CD73, CD105, CD117, CD200, OCT-4 and/or HLA-G.

In another embodiment, hematopoietic cells, e.g., CD34⁺, CD133⁺, KDR⁺ orThy-1⁺ cells, are sorted, e.g., isolated, on the basis of markerscharacteristic of undifferentiated hematopoietic cells. Such sorting canbe done, e.g., in a population of cells that has not been sorted, e.g.,a population of cells from a perfusion or a tissue digestion, whereinCD34⁺ cells represent a minority of the cells present in the population.Such sorting can also be done in a population of cells that is mostly(e.g., greater than 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%)hematopoietic cells as, for example, a purification step. For example,in a specific embodiment, CD34⁺ cells, KDR⁺ cells, Thy-1⁺ cells, and/orCD133⁺ cells are retained during sorting to produce a population ofundifferentiated hematopoietic cells.

In another embodiment, cells, e.g., hematopoietic cells are sorted,e.g., excluded, on the basis of markers of lineage-differentiated cells.For example, cells, in a population of hematopoietic cells, that areCD2⁺, CD3⁺, CD11b⁺, CD11c⁺, CD 14⁺, CD 16⁺, CD 19⁺, CD24⁺, CD56⁺, CD66b⁺and/or glycophorin A⁺ are excluded during sorting from the population ofhematopoietic cells to produce a population of undifferentiatedhematopoietic cells.

In another embodiment, hematopoietic cells can be sorted, e.g.,isolated, on the basis of lack of expression of, e.g., lineage markers.In a specific embodiment, for example, hematopoietic cells, e.g., CD34⁺cells, can be isolated based on a determination that the cells are oneor more of CD2⁻, CD3⁻, CD11b⁻, CD11c⁻, CD 14⁻, CD 16⁻, CD 19⁻, CD24⁻,CD56⁻, CD66b⁻ and/or glycophorin A⁻.

In another embodiment, magnetic beads can be used to separate cells,e.g., DYNABEADS® (Invitrogen). The cells may be sorted using a magneticactivated cell sorting (MACS) technique, a method for separatingparticles based on their ability to bind magnetic beads (0.5-100 μmdiameter). A variety of useful modifications can be performed on themagnetic microspheres, including covalent addition of antibody thatspecifically recognizes a particular cell surface molecule or hapten.The beads are then mixed with the cells to allow binding. Cells are thenpassed through a magnetic field to separate out cells having thespecific cell surface marker. In one embodiment, these cells can thenisolated and re-mixed with magnetic beads coupled to an antibody againstadditional cell surface markers. The cells are again passed through amagnetic field, isolating cells that bound both the antibodies. Suchcells can then be diluted into separate dishes, such as microtiterdishes for clonal isolation.

In another embodiment, placental stem cells, e.g., placentalhematopoietic cells or adherent placental stem cells, can be identifiedand characterized by a colony forming unit assay. Colony forming unitassays are commonly known in the art, such as MESENCULT™ medium (StemCell Technologies, Inc., Vancouver British Columbia).

Placental stem cells can be assessed for viability, proliferationpotential, and longevity using standard techniques known in the art,such as trypan blue exclusion assay, fluorescein diacetate uptake assay,propidium iodide uptake assay (to assess viability); and thymidineuptake assay, MTT cell proliferation assay (to assess proliferation).Longevity may be determined by methods well known in the art, such as bydetermining the maximum number of population doubling in an extendedculture.

Placental stem cells can also be separated from other placental cellsusing other techniques known in the art, e.g., selective growth ofdesired cells (positive selection), selective destruction of unwantedcells (negative selection); separation based upon differential cellagglutinability in the mixed population as, for example, with soybeanagglutinin; freeze-thaw procedures; filtration; conventional and zonalcentrifugation; centrifugal elutriation (counter-streamingcentrifugation); unit gravity separation; countercurrent distribution;electrophoresis; and the like.

5.2. Expansion of Hematopoietic Cells

Once a population of hematopoietic cells is obtained, the population isexpanded. One unit of erythrocytes is expected to comprise about1-2×10¹² red blood cells. Hematopoietic stem cell population doublingrequires approximately 36 hours. Thus, starting from about 5×10⁷hematopoietic cells according to standard methods, and assuming 100%efficiency in expansion and differentiation, production of a unit oferythrocytes would require approximately 14 hematopoietic cellpopulation doublings, or approximately 3 weeks. The method described indetail below improves on standard methods by improving the cultureconditions of hematopoietic cells and increasing the number ofhematopoietic cells during expansion per unit time.

5.2.1. Shortened Hematopoietic Cell Expansion Time

Cells, including hematopoietic cells, comprise cell cycle controlmechanisms, which include cyclins and cyclin-dependent kinases (CDKs),that control the rate of cell division. Cell cycle checkpoints are usedby cells to monitor and regulate the progress of the cell cycle. If acell fails to meet the requirements of a phase it will not be allowed toproceed to the next phase until the requirements have been met. Theprocesses associated with qualifying the cell for progression throughthe different phases of the cell cycle (checkpoint regulation) arerelatively slow and contribute to the relatively modest rate of celldivision observed in mammalian cells, even under optimal in vitroculture conditions.

In one embodiment of the method of producing erythrocytes, the methoduses hematopoietic cells that have a reduced population doubling time.In a specific embodiment, the hematopoietic cells are modified toexpress higher-than-normal levels of a cell cycle activator, or alower-than-normal level of a cell cycle inhibitor, wherein theengineered cells have a detectably shorter doubling time than unmodifiedhematopoietic cells. In a more specific embodiment, the hematopoieticcells are modified to express a higher-than-normal level of one or moreof the cell cycle activator cyclin T2 (CCNT2), cyclin T2B (CCNT2B),CDC7L1, CCN1, cyclin G (CCNG2), cyclin H(CCNH), CDKN2C, CDKN2D, CDK4,cyclin D1, cyclin A, cyclin B, Hes1, Hox genes and/or FoxO.

In another more specific embodiment, the hematopoietic cells express alower-than-normal level of CDK inhibitors p21, p27 and/or TReP-132.Reduction of expression of CDK inhibitors can be accomplished by anymeans known in the art, e.g., the use of small molecule inhibitors,antisense oligonucleotides targeted to a p21, p27 and/or TReP-132 DNA,pre-mRNA or mRNA sequence, RNAi, or the like.

Modifications of hematopoietic progenitor cells in the context of thepresent method of producing erythrocytes are expected to be safe in atherapeutic context, as erythrocytes are enucleated and incapable ofreplication.

In another specific embodiment, the hematopoietic cells are modified toexpress higher-than-normal levels of a cell cycle activator, wherein theengineered cells have a detectably shorter doubling time than, ordetectably increased rate of proliferation compared to, unmodifiedhematopoietic cells, and where the increased expression of a cell cycleactivator is inducible. Any inducible promoter known in the art can beused to construct such a modified hematopoietic cell, e.g., atetracycline-inducible gene expression system using a stably expressedreverse tetracycline-controlled transactivator (rtTA) under the controlof a CMV promoter (e.g., REVTET-ON® System, Clontech Laboratories, PaloAlto, Calif.); U.S. Patent Application Publication No. 2007/0166366“Autologous Upregulation Mechanism Allowing Optimized Cell Type-Specificand Regulated Gene Expression Cells”; and U.S. Patent ApplicationPublication No. 2007/0122880 “Vector for the Inducible Expression ofGene Sequences,” the disclosure of each of which is incorporated hereinby reference in its entirety.

Expression of a gene encoding a cell cycle inhibitor or negative cellcycle regulator can be disrupted in a hematopoietic cell, e.g., byhomologous or non-homologous recombination using standard methods.Disruption of expression of a cell cycle inhibitor or negative cellcycle regulator can also be effected, e.g., using an antisense moleculeto, e.g., p21, p27 and/or TReP-132.

In another embodiment, hematopoietic cells used to produce erythrocytesare modified to express notch 1 ligand such that expression of the notch1 ligand results in detectably decreased senescence of the hematopoieticcells compared to unmodified hematopoietic cells; see Berstein et al.,U.S. Patent Application Publication 2004/0067583 “Methods forImmortalizing Cells,” the disclosure of which is incorporated herein byreference in its entirety.

In another specific embodiment, the medium in which the hematopoieticcells are expanded enhance faithful DNA replication, e.g., the mediumincludes one or more antioxidants.

In a preferred embodiment, the method of producing erythrocytes includesa step that excludes any modified hematopoietic cells, orpre-erythrocyte precursors, from the final population of isolatederythrocytes produced in the method disclosed herein. Such separationcan be accomplished as described elsewhere herein on the basis of one ormore markers characteristic of hematopoietic cells not fullydifferentiated into erythrocytes. The exclusion step can be performedsubsequent to an isolation step in which erythrocytes are selected onthe basis of erythrocyte-specific markers, e.g., CD36 and/or glycophorinA.

5.2.2. Feeder Cell-Independent Expansion and Differentiation ofHematopoietic Cells

In certain embodiments, hematopoietic cells, e.g., stem cells orprogenitor cells, used in the methods provided herein are expanded anddifferentiated in culture without the use of a feeder layer. Culture ofthe hematopoietic cells as provided herein results in continuousexpansion of the hematopoietic cells and differentiation of erythrocytesfrom said cells.

Feeder cell-independent expansion and differentiation of hematopoieticcells can take place in any container compatible with cell culture andexpansion, e.g., flask, tube, beaker, dish, multiwell plate, bag or thelike. In a specific embodiment, feeder cell-independent expansion ofhematopoietic cells takes place in a bag, e.g., a flexible,gas-permeable fluorocarbon culture bag (for example, from AmericanFluoroseal). In a specific embodiment, the container in which thehematopoietic cells are expanded is suitable for shipping, e.g., to asite such as a hospital or military zone wherein the expandedhematopoietic cells are further expanded and differentiated, e.g., usingthe bioreactor described below.

In certain embodiments, hematopoietic cells, in certain embodiments, areexpanded and differentiated, in continuous fashion, in a culture mediumcomprising stem cell factor (SCF), erythropoietin (Epo), andinterleukin-3 (IL-3).

Thus, in one aspect, provided herein is a method of producingerythrocytes, comprising expanding and differentiating a population ofhematopoietic cells in a medium in the absence of feeder cells, whereina plurality of hematopoietic cells within said population ofhematopoietic cells differentiate into erythrocytes during saidexpanding; and isolating said erythrocytes from said medium, whereinsaid medium comprises SCF at a concentration of about 10 to about 10,000ng/mL, IL-3 at a concentration of about 0.01 to about 500 ng/mL, and EPOat a concentration of about 0.1 to about 10 IU/mL, and wherein said SCF,IL-3 and Epo are not comprised within an undefined component of saidmedium (e.g., serum). In a specific embodiment of the method, saidmedium does not comprise one or more, or any, of Flt-3L, IL-11,thrombopoietin (Tpo), homeobox-B4 (HoxB4), or methylcellulose. In otherspecific embodiments, said medium comprises SCF at a concentration ofabout 20 to about 2000 ng/mL; about 50 to about 1000 ng/mL; or about 100ng/mL. In other specific embodiments, said medium comprises IL-3 at aconcentration of about 0.1 to about 100 ng/mL; about 1 to about 50ng/mL; or about 5 ng/mL. In other specific embodiments, said mediumcomprises EPO at a concentration of about 1 to about 5 IU/mL; or about 2to about 3 IU/mL.

In certain embodiments, the medium facilitates the expansion ofhematopoietic stem cells in culture, e.g., CD34⁻ hematopoietic stemcells, wherein the cells are seeded at 5×10⁵ cells/mL or less, 2.75×10⁴cells/mL or less, or 5×10⁴ cells/mL or less; wherein Epo is present(e.g., the medium comprises) 5 IU/ml or less, 3 IU/mL or less, or 1IU/mL or less; and SCF is present (e.g., the medium comprises) at 1ng/mL or more, 50 ng/mL or more, or 100 ng/mL or more. In a specificembodiment, the cells are seeded at 5×10⁴ cells/mL or less, and themedium comprises 1 IU/mL or less Epo and 100 ng/mL or more SCF.

In another specific embodiment of the method, said medium furthercomprises insulin-like growth factor 1 (IGF-1) at a concentration ofabout 1 to about 1000 ng/mL and lipids at a concentration of about 1 toabout 1000 μg/mL, wherein said lipids comprise a mixture of protein andcholesterol (e.g., Lipids Cholesterol enriched from adult bovine serum;Cat. No. C7305-1G, Sigma, St Louis, Mo.); and wherein said mediumcomprises hydrocortisone at a concentration of about 0.01 to about 100μM, or dexamethasone at a concentration of about 0.01 μM to about 100μM. In more specific embodiments, said medium comprises IGF-1 at aconcentration of about 10 to about 500 ng/mL; or about 20 to about 100ng/mL. In other more specific embodiments, said medium comprises lipidsat a concentration of about 10 to about 500 ng/mL; or about 20 to about100 ng/mL. In other more specific embodiments, said medium compriseshydrocortisone at a concentration of about 0.1 to about 50 μM; or about0.5 to about 10 μM. In other more specific embodiments, said mediumcomprises dexamethasone at a concentration of about 0.05 to about 20 μM;or about 0.1 to about 10 μM.

In a more specific embodiment of the method, the medium comprises about100 ng/mL SCF, about 3 U/mL Epo, about 40 ng/mL IGF-1, about 5 ng/mLIL-3, about 1 μM Dexamethasone, and 40 μg/ml lipids, wherein said lipidscomprise a mixture of protein and cholesterol. In another more specificembodiment of the method, the medium comprises about 100 ng/mL SCF,about 2 U/mL Epo, about 40 ng/mL IGF-1, about 5 ng/mL IL-3, about 1 μMhydrocortisone, and 50 ng/ml lipids, wherein said lipids comprise amixture of protein and cholesterol.

In certain other embodiments, hematopoietic cells, in certainembodiments, are expanded and differentiated, in continuous fashion, ina culture medium comprising SCF; Epo; IGF-1; lipids, wherein the lipidscomprise a mixture of proteins and cholesterol (e.g., Lipids CholesterolRich from adult bovine serum; Cat. No. C7305-1G, Sigma, St Louis, Mo.);and either hydrocortisone or dexamethasone. In specific embodiments,said medium comprises SCF at a concentration of about 10 to about 10,000ng/mL; about 20 to about 2000 ng/mL; about 50 to about 1000 ng/mL; about100 ng/mL; or about 100 ng/mL. In other specific embodiments, saidmedium comprises Epo at a concentration of about 1 to about 5 IU/mL; orabout 2 to about 3 IU/mL. In other specific embodiments, said mediumcomprises IGF-1 at a concentration of about 1 to about 1000 ng/mL; about10 to about 500 ng/mL; about 20 to about 100 ng/mL; or about 40 ng/mL.In other specific embodiments, said medium comprises said lipids at aconcentration of about 1 to about 1000 μg/mL; about 10 to about 500ng/mL; about 20 to about 100 ng/mL; or about 40 ng/mL. In other specificembodiments, said medium comprises hydrocortisone at a concentration ofabout 0.1 μM to about 10 μM; about 0.5 UV to about 5 μM; or about 1 μM.In other specific embodiments, said medium comprises dexamethasone at aconcentration of about 0.1 μM to about 10 μM; about 0.5 μM to about 5μM; or about 1 μM.

In addition to the method, provided herein are any of the mediadescribed above as compositions. In certain embodiments of any of themethods or compositions provided herein, the medium can be a serum-freemedium, e.g., STEMSPAN® (Cat. No. 09650, Stem Cell Technologies,Vancouver, Canada).

In another embodiment, hematopoietic cells are expanded by culturingsaid cells in contact with an immunomodulatory compound, e.g., a TNF-αinhibitory compound, for a time and in an amount sufficient to cause adetectable increase in the proliferation of the hematopoietic cells overa given time, compared to an equivalent number of hematopoietic cellsnot contacted with the immunomodulatory compound. See, e.g., U.S. PatentApplication Publication No. 2003/0235909, the disclosure of which ishereby incorporated by reference in its entirety. In a preferredembodiment, the immunomodulatory compound is3-(4-amino-1-oxo-1,3-dihydroisoindol-2-yl)-piperidine-2,6-dione; 3-(4′aminoisolindoline-1′-one)-1-piperidine-2,6-dione;4-(amino)-2-(2,6-dioxo(3-piperidyl))isoindoline-1,3-dione;4-amino-2-[(3RS)-2,6-dioxopiperidin-3-yl]-2H-isoindole-1,3-dione;α-(3-aminophthalimido) glutarimide; pomalidomide, lenalidomide, orthalidomide. In another embodiment, said immunomodulatory compound is acompound having the structure

wherein one of X and Y is C═O, the other of X and Y is C═O or CH₂, andR² is hydrogen or lower alkyl, or a pharmaceutically acceptable salt,hydrate, solvate, clathrate, enantiomer, diastereomer, racemate, ormixture of stereoisomers thereof. In another embodiment, saidimmunomodulatory compound is a compound having the structure

wherein one of X and Y is C═O and the other is CH₂ or C═O;

R¹ is H, (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl,(C₀-C₄)alkyl-(C₂-C₅)heteroaryl, C(O)R³, C(S)R³, C(O)OR⁴,(C₁-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵, C(O)NHR³,C(S)NHR³, C(O)NR³R^(3′), C(S)NR³R^(3′) or (C₁-C₈)alkyl-O(CO)R⁵;

R² is H, F, benzyl, (C₁-C₈)alkyl, (C₂-C₈)alkenyl, or (C₂-C₈)alkynyl;

R³ and R^(3′) are independently (C₁-C₈)alkyl, (C₃-C₇)cycloalkyl,(C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl,(C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, (C₀-C₄)alkyl-(C₂-C₅)heteroaryl,(C₀-C₈)alkyl-N(R⁶)₂, (C₁-C₈)alkyl-OR⁵, (C₁-C₈)alkyl-C(O)OR⁵,(C₁-C₈)alkyl-O(CO)R⁵, or C(O)OR⁵;

R⁴ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, (C₁-C₄)alkyl-OR⁵,benzyl, aryl, (C₀-C₄)alkyl-(C₁-C₆)heterocycloalkyl, or(C₀-C₄)alkyl-(C₂-C₅)heteroaryl;

R⁵ is (C₁-C₈)alkyl, (C₂-C₈)alkenyl, (C₂-C₈)alkynyl, benzyl, aryl, or(C₂-C₅)heteroaryl;

each occurrence of R⁶ is independently H, (C₁-C₈)alkyl, (C₂-C₈)alkenyl,(C₂-C₈)alkynyl, benzyl, aryl, (C₂-C₅)heteroaryl, or(C₀-C₈)alkyl-C(O)O—R⁵ or the R⁶ groups can join to form aheterocycloalkyl group;

n is 0 or 1; and

* represents a chiral-carbon center;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.In another embodiment, said immunomodulatory compound is a compoundhaving the structure

wherein:

one of X and Y is C═O and the other is CH₂ or C═O;

R is H or CH₂OCOR′;

(i) each of R¹, R², R³, or R⁴, independently of the others, is halo,alkyl of 1 to 4 carbon atoms, or alkoxy of 1 to 4 carbon atoms or (ii)one of R¹, R², R³, or R⁴ is nitro or —NHR⁵ and the remaining of R¹, R²,R³, or R⁴ are hydrogen;

R⁵ is hydrogen or alkyl of 1 to 8 carbons

R⁶ hydrogen, alkyl of 1 to 8 carbon atoms, benzo, chloro, or fluoro;

R′ is R⁷—CHR¹⁰—N(R⁸R⁹);

R⁷ is m-phenylene or p-phenylene or —(C_(n)H_(2n))— in which n has avalue of 0 to 4;

each of R⁸ and R⁹ taken independently of the other is hydrogen or alkylof 1 to 8 carbon atoms, or R⁸ and R⁹ taken together are tetramethylene,pentamethylene, hexamethylene, or —CH₂CH₂X₁CH₂CH₂— in which X₁ is —O—,—S—, or —NH—;

R¹⁰ is hydrogen, alkyl of to 8 carbon atoms, or phenyl; and

* represents a chiral-carbon center;

or a pharmaceutically acceptable salt, hydrate, solvate, clathrate,enantiomer, diastereomer, racemate, or mixture of stereoisomers thereof.

In a specific embodiment, expansion of hematopoietic cells is performedin IMDM supplemented with 20% BITS (BSA, recombinant human insulin andtransferrin), SCF, Flt-3 ligand, IL-3, and4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione (10 μM in0.05% DMSO). In a more specific embodiment, about 5×10⁷ hematopoieticcells, e.g., CD34⁺ cells, are expanded in the medium to from about5×10¹⁰ cells to about 5×10¹² cells, which are resuspended in 100 mL ofIMDM to produce a population of expanded hematopoietic cells. Thepopulation of expanded hematopoietic cells is preferably cryopreservedto facilitate shipping.

Production of erythrocytes by the methods and in the media describedabove, is preferably performed in a bioreactor, e.g., the bioreactorexemplified elsewhere herein.

In various specific embodiments, at least 50%, 55%, 60%, 65%, 70%. 75%,80%, 85%, 90%, 95%, 97%, 98%, or 99% of the hematopoietic cells aredifferentiated to erythrocytes and/or polychromatophilic erythrocytes.

In one embodiment, differentiation of hematopoietic cells, e.g., theexpanded hematopoietic cells described above, can be accomplished byculturing said cells in contact with an immunomodulatory compound, e.g.,a TNF-α inhibitory compound as described above, for a time and in anamount sufficient to cause a detectable increase in the proliferation ofthe hematopoietic cells over a given time, compared to an equivalentnumber of hematopoietic cells not contacted with the immunomodulatorycompound. See, e.g., U.S. Patent Application Publication No.2003/0235909, the disclosure of which is incorporated herein byreference in its entirety.

In certain embodiments, the method of expansion and differentiation ofthe hematopoietic cells, as described herein, comprises maintaining thecell population comprising said hematopoietic cells is maintained atbetween about 2×10⁴ and about 2×10⁵ cells per milliliter duringexpansion and differentiation. In certain other embodiments, the methodof expansion and differentiation of the hematopoietic cells, asdescribed herein, comprises maintaining the cell population comprisingsaid hematopoietic cells is maintained at no more than about 1×10⁵ cellsper milliliter.

Differentiation of the hematopoietic cells into erythrocytes can beassessed by detecting erythrocyte-specific markers, e.g., by flowcytometry. Erythrocyte-specific markers include, but are not limited to,CD36 and glycophorin A. Differentiation can also be assessed by visualinspection of the cells under a microscope. The presence of typicalbiconcave cells confirms the presence of erythrocytes. The presence oferythrocytes (including reticulocytes) can be confirmed using a stainfor deoxyribonucleic acid (DNA), such as Hoechst 33342, TO-PRO®-3, DRAG5or the like. Nucleated precursors to erythrocytes typically stainpositive with a DNA-detecting stain, while erythrocytes andreticulocytes are typically negative. Differentiation of hematopoieticcells to erythrocytes can also be assessed by progressive loss oftransferring receptor (CD71) expression and/or laser dye styryl stainingduring differentiation. Erythrocytes can also be tested fordeformability using, e.g., an ektacytometer or diffractometer. See,e.g., Bessis M and Mohandas N, “A Diffractometric Method for theMeasurement of Cellular Deformability,” Blood Cells 1:307 (1975);Mohandas N. et al., “Analysis of Factors Regulating ErythrocyteDeformability,” J. Clin. Invest. 66:563 (1980); Groner W et al., “NewOptical Technique for Measuring Erythrocyte Deformability with theEktacytometer,” Clin. Chem. 26:1435 (1980). Fully-differentiatederythrocytes have a mean corpuscular volume (MCV) of about 80 to about108 fL (femtoliters); mean corpuscular hemoglobin (MCH) of about 17 toabout 31 pg, and a mean corpuscular hemoglobin concentration (MCHC) ofabout 23% to about 36%.

The time for differentiation of hematopoietic cells into erythrocytescan be from about 3 days to about 120 days. In one embodiment, thedifferentiation time is about 7 days to about 35 days. In anotherembodiment, the differentiation time is about 14 days to about 28 days.

5.3. Separation of Erythrocytes From Precursors

Erythrocytes produced by the methods described above are preferablyseparated from hematopoietic cells, and, in certain embodiments, fromprecursors of erythrocytes. Such separation can be effected, e.g., usingantibodies to CD36 and/or glycophorin A. Separation can be achieved byknown methods, e.g., antibody-mediated magnetic bead separation,fluorescence-activated cell sorting, passage of the cells across asurface or column comprising antibodies to CD36 and/or glycophorin A, orthe like. In another embodiment, erythrocyte separation is achieved bydeoxygenating the culture medium comprising the erythrocytes, followedby magnetic separation of deoxygenated erythrocytes from other cells.

Erythrocytes can be continuously separated from a population of cells,e.g., from a second expanded hematopoietic cell population as describedabove, or can be separated at intervals. In certain embodiments, forexample, isolation of erythrocytes is performed, e.g., every 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes, orevery 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0,8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23 or 24 hours, or more, or when one or more culturecondition criteria are met, e.g., achievement in the culture of aparticular cell density; achievement in the culture of a particularnumber of cells per milliliter expressing certain erythrocyte markers,e.g., CD36 or glycophorin A; or the like. Separation of erythrocytesfrom a cell population is preferably performed using a bioreactor, asdescribed below.

5.4. Bioreactor Production of Erythrocytes

In another aspect of the method of producing erythrocytes, hematopoieticcells are expanded and differentiated in a bioreactor in the absence offeeder cells. The bioreactor in which the hematopoietic cells aredifferentiated can be the same bioreactor in which the hematopoieticcells are expanded, or can be a separate bioreactor. In anotherembodiment, the bioreactor is constructed to facilitate expansion of thehematopoietic cells entirely in the bioreactor. In another embodiment,the bioreactor is constructed to allow expansion of hematopoietic cellswithout feeder cells.

In another embodiment, the bioreactor is constructed to allow continuousflow of cells in media, enabling the continuous separation ofdifferentiated erythrocytes from remaining cells in the bioreactor. Thecontinuous flow and cell separation allows for the bioreactor to beconstructed in a substantially smaller volume than would bioreactorsusing batch methods of producing erythrocytes. In another embodiment,the bioreactor is constructed to allow periodic, e.g., non-continuousflow of cells in media, enabling the periodic separation ofdifferentiated erythrocytes from remaining cells in the bioreactor. Theperiodic flow and cell separation preferably allows for the bioreactorto be constructed in a substantially smaller volume than wouldbioreactors using batch methods of producing erythrocytes. In specificembodiments, isolation of erythrocytes is performed, e.g., every 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60minutes, or every 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0,7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23 or 24 hours, or more. In another specificembodiment, isolation of erythrocytes is performed periodically when oneor more culture condition criteria are met, e.g., achievement in theculture of a particular cell density; achievement in the culture of aparticular number of cells per milliliter expressing certain erythrocytemarkers, e.g., CD36 or glycophorin A; or the like.

In certain embodiments, the bioreactor is disposable.

In one embodiment, the bioreactor comprises a culturing element and acell separation element. In another embodiment, the bioreactor comprisesa medium gas provision element. In another embodiment, the bioreactorcomprises a cell factor element comprising one or more bioactivecompounds. In another embodiment, the elements of the bioreactor aremodular; e.g., separable from each other and/or independently usable. Inone embodiment, the capacity of the bioreactor is about 100 mL, 200 mL,300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, or about 900 mL, orabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40,45 or 50 liters. In another embodiment, the bioreactor, including allcomponents, occupies about 47 cubic feet or less. In another embodiment,the bioreactor is capable of culturing up to about 10¹⁰, 10¹¹, or about10¹² cells, e.g., hematopoietic cells.

In one embodiment, the culturing element comprises a compartment able toreceive culture medium, e.g., culture medium comprising hematopoieticcells. The culturing element comprises a port that allows for theintroduction of media and/or hematopoietic cells for culture. Such aport can be any art-acceptable port for such devices, e.g., a Luer-lockseal port. The culturing element also comprises one or more ports forthe passage of media to the cell separation element. The culturingelement optionally further comprises a port for the introduction ofbioactive compounds into the interior of the culturing element, e.g., aport that facilitates connection of the cell factor element to theculturing element. In a specific embodiment, hematopoietic cells,including differentiating hematopoietic cells, in the culturing elementare continuously circulated in medium to a cell separation element (seebelow) to isolate erythrocytes and/or polychromatophilic erythrocytesand/or other erythrocyte precursors.

The culturing element, in a specific embodiment, comprises a pluralityof interior surfaces or structures, e.g., tubes, cylinders, hollowfibers, a porous substrate, or the like. The surfaces can be constructedof any material suitable for the culture of cells, e.g., tissue cultureplastic, flexible pharmaceutical grade plastic, hydroxyapatite,polylactic acid (PLA), polyglycolic acid copolymer (PLGA), polyurethane,polyhydroxyethyl methacrylate, or the like. Hollow fibers typicallyrange from about 100 μm to about 1000 μm in diameter, and typicallycomprise pores that allow passage of molecules no more than about 5 kDa,10 kDa, 15 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa,90 kDa, 100 kDa, 125 kDa, 150 kDa, 175 kDa, 200 kDa, 150 kDa, 300 kDa,350 kDa, 400 kDa, 450 or 500 kDa.

The cell separation element comprises at least one port for receivingmedium, comprising cells, from the culturing element. The cellseparation element comprises one or more components that facilitate orenable the separation of at least one type of cell, e.g., erythrocytes,from cells in medium from the culture element. Such separation can beeffected, e.g., using antibodies to CD36 and/or glycophorin A.Separation can be achieved by known methods, e.g., antibody-mediatedmagnetic bead separation, fluorescence-activated cell sorting, passageof the cells across a surface or column comprising antibodies to CD36and/or glycophorin A, or the like. Separation can also be achieved basedon cell size or cell density. In a specific embodiment, the cellseparation element is connected to the cell culturing element, andmedium comprising hematopoietic cells, differentiating hematopoieticcells and erythrocytes is continually passed through the cell separationelement so as to continually remove cells, e.g., erythrocytes from themedium.

In another embodiment, erythrocyte separation is achieved bydeoxygenating culture medium comprising the erythrocytes, followed bymagnetic attraction of deoxygenated erythrocytes, e.g., to a surface orother point of collection.

In another embodiment, the bioreactor comprises a cell separationelement. The cell separation element can comprise one or more componentsthat enable the separation of one or more non-erythrocytic cells (e.g.,undifferentiated or non-terminally differentiated hematopoietic cells)from erythrocytes in the medium. In certain embodiments, the cellseparation element is able to calculate an approximate number oferythrocytes generated, or is able to alert a user that a sufficientnumber of erythrocytes has been generated to constitute a unit,according to preset user parameters.

The bioreactor, in another embodiment, further comprises a gas provisionelement that provides appropriate gases to the culture environment,e.g., contacts the culture medium with a mixture of 80% air, 15% O₂ and5% CO₂, 5% CO₂ in air, or the like. In another embodiment, thebioreactor comprises a temperature element that maintains the medium,the bioreactor, or both at a substantially constant temperature, e.g.,about 35° C. to about 39° C., or about 37° C. In another embodiment, thebioreactor comprises a pH monitoring element that maintains the mediumat a constant pH, e.g., about pH 7.2 to about pH 7.6, or about pH 7.4.In specific embodiments, the temperature element and/or pH monitoringelement comprises a warning that activates when temperature and/or pHexceed or fall below set parameters. In other specific embodiments, thetemperature element and/or pH monitoring element are capable ofcorrecting out-of-range temperature and/or pH.

In a specific embodiment, the bioreactor comprises a cell separationelement and a gas provision element that provides gases to the cultureenvironment, whereby the gas provision element enables the partial orcomplete deoxygenation of erythrocytes, enabling erythrocyte separationbased on the magnetic properties of the hemoglobin contained therein. Ina more specific aspect, the bioreactor comprises an element that allowsfor the regular, or iterative, deoxygenation of erythrocytes produced inthe bioreactor, to facilitate magnetic collection of the erythrocytes.

In another embodiment, the function of the bioreactor is automated,e.g., controlled by a computer. The computer can be, for example, adesktop personal computer, a laptop computer, a Handspring, PALM® orsimilar handheld device; a minicomputer, mainframe computer, or thelike.

5.5. Erythrocyte Units Produced From Hematopoietic Cells

Erythrocyte units produced according to the methods detailed above cancomprise erythrocytes in any useful number or combination of geneticbackgrounds.

In various embodiments, erythrocyte units produced by the methodsprovided herein comprise at least about, at most about, or about 1×10⁸,5×10⁸, 1×10⁹, 5×10⁹, 1×10¹⁰, 5×10¹⁰, 1×10¹¹, 5×10¹¹ or 1×10¹²erythrocytes. In various other embodiments, the erythrocyte unitscomprise at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 98% or 99% completely-differentiated erythrocytes. In various otherembodiments, the erythrocyte units comprise less than 60%, 50%, 40%,30%, 20%, 10%, 5%, 2% or 1% erythrocyte precursors of any kind. Incertain embodiments, the erythrocyte units produced by the methodsdescribed herein comprise less than about 60%, about 50%, about 40%,about 30%, about 20%, about 19%, about 18%, about 17%, about 16%, about15%, about 14%, about 14%, about 12%, about 11%, about 10%, about 9%,about 8%, about 7%, about 6% or about 5% reticulocytes, or othernon-erythrocytic hematopoietic cells. In another embodiment, the unitcomprises erythrocytes from hematopoietic cells from a singleindividual. In another embodiment, the unit comprises erythrocytesdifferentiated from hematopoietic cells from a plurality of individuals.In another embodiment, the unit comprises erythrocytes fromhematopoietic cells from matched human placental perfusate and cordblood. In another embodiment, substantially all (e.g., greater than 99%)of the erythrocytes in a unit of erythrocytes are type O. In anotherembodiment, substantially all (e.g., greater than 99%) of theerythrocytes in a unit of erythrocytes are type A. In anotherembodiment, substantially all (e.g., greater than 99%) of theerythrocytes in a unit of erythrocytes are type B. In anotherembodiment, substantially all (e.g., greater than 99%) of theerythrocytes in a unit of erythrocytes are type AB. In anotherembodiment, substantially all (e.g., greater than 99%) of theerythrocytes in a unit of erythrocytes are Rh positive. In anotherembodiment, substantially all (e.g., greater than 99%) of theerythrocytes in a unit of erythrocytes are Rh negative.

Naturally-occurring erythrocytes possess certain characteristics thatallow the flow of blood through capillaries. For example, erythrocytesin the aggregate produce non-Newtonian flow behavior, e.g., theviscosity of blood is highly dependent upon shear rates. Normalerythrocytes are deformable and able to build up aggregates/rouleaux.The deformability of erythrocytes appears to be related to theirlifespan in the blood, about 100-120 days; removal of erythrocytes fromthe blood appears to be related to loss of deformability. Normalaggregability of erythrocytes facilitates the cells' flow through thecapillaries, while abnormally increased or decreased aggregabilitydecreases flow. Thus, in preferred embodiments, units of erythrocytesproduced by the methods disclosed herein are assayed as a part ofquality control, e.g., for one or more characteristics ofnaturally-occurring erythrocytes. In certain embodiments, samples oferythrocytes produced by the methods disclosed herein are suspended innatural or artificial plasma and tested for one or more of viscosity,viscoelasticity, relaxation time, deformability, aggregability,blood/erythrocyte suspension yield stress, and mechanical fragility,using normal blood or normal erythrocytes as a control or comparator. Incertain other embodiments, samples of erythrocytes produced as describedherein are assayed for oxygen carrying capacity and oxygen releasecapacity, using normal blood or an equivalent number ofnaturally-occurring erythrocytes as a control.

6. EXAMPLES 6.1. Example 1 Characterization of CD34⁺ Cells from HumanPlacental Perfusate (HPP) and Umbilical Cord Blood (UCB)

Umbilical cord blood (UCB) was removed from postpartum placentas underinformed consent. The exsanguinated placentas were then perfused togenerate HPP, as described in U.S. Pat. No. 7,045,148, the disclosure ofwhich is incorporated herein by reference in its entirety. After removalof red blood cells (RBCs) the total nucleated cells (TNCs) werecollected and frozen. This method typically resulted in the collectionof about 1-2.5×10⁹ TNCs, compared to around 500 million TNC isolatedfrom UCB.

Flow cytometric analysis of the TNC isolated from exsanguinatedplacentas indicates a high percentage of CD34⁺ cell population ascompared to conventional umbilical cord blood (UCB) generated cellularproduct. TNC from HPP, collected as above, contains about 2% to 6% CD34⁺cells, compared to about 0.3% to 1% of the TNC in UCB.

The flow cytometric analysis of the TNC isolated from HPP indicates thata high percentage of the CD34⁺ cell population is CD45⁻ (FIG. 1).

CD34⁺ cells from HPP were plated in a colony-forming unit assay, and theratio (%) of the burst forming unit-erythroid (BFU-E) to the colonyforming unit-erythroid (CFU-E) was determined, as well as the number ofcolony-forming unit-granulocyte, macrophage (CFU-GM) and the number ofcolony-forming unit-granulocyte, erythrocyte, monocyte (CFU-GEMM) (Table1). The clonogenicity was also assessed (Table 1).

TABLE 1 BFU-E/ Sample Cell Purity CFU-E CFU-GM CFU-GEMM ClonogenicityDonor 1 88% 50.1% 49.5% 0.4% 23.1% Donor 2 92% 54.1% 44.1% 1.7% 26.1%Donor 3 94% 32.7% 60.6% 6.7% 19.7%

The colony-forming unit assay was performed according to themanufacturer's protocol (StemCell Technologies, Inc.). In brief, CD34⁺cell suspensions were placed into a methylcellulose medium supplementedwith stem cell factor (SCF), granulocyte colony-stimulating factor(G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF),interleukin 3 (IL-3), interleukin 6 (IL-6) and erythropoietin (Epo) at100 cells/plate, 300 cells/plate and 1000 cells/plate. For each celldensity, a triplicate assay was performed followed by incubation for 2to 3 weeks. Colony evaluation and enumeration were performed using lightmicroscopy.

In a separate experiment, the ratio (BFU-E)/(CFU-E) for CD34⁺ cells fromHPP and UCB was 46% and 30%, respectively (based on the average valuefor three donors).

These results suggest that HPP-derived cells contain a higher number ofCD34⁺ cells with increased erythrogenic activity relative to UCB-derivedstem cells.

6.2. Example 2 Recovery of Hematopoietic Stem Cells (HSCs)

HPP and UCB cells were generally purified as described in Example 1using either Ficoll, hetastarch or ammonium chloride to obtain totalnucleated cells (TNCs). TNCs were then used in a positive selectionprocedure to isolate CD34⁺ cells using anti-CD34 beads and RoboSepfollowing the protocol provided by the manufacturer (StemCellTechnologies, Inc.) In this experiment, CD34⁺ cells were isolated withgreater than 90% purity (FIG. 2). Alternatively, EASYSEP® HumanProgenitor Cell Enrichment Kit (StemCell Technologies, Inc.) was used ina negative selection procedure to deplete the lineage committed cells byusing Human Progenitor Cell Enrichment Cocktail with monoclonalantibodies to the following human cell surface antigens: CD2, CD3,CD11b, CD11c, CD14, CD16, CD19, CD24, CD56, CD66b, Glycophorin A. Usingthe negative selection process, 90% CD34⁺ cells were recovered from theraw materials; the cell composition of the recovered HSCs is summarizedin Table 2.

A colony-forming unit assay of the isolated CD34⁺ cells showed that thecolony forming frequency of negative selection HSCs is comparable topositive selection HSCs and the BFU-E forming frequency of negativeselection HSCs is higher than that of positive selection HSCs.

TABLE 2 Cell composition of enriched HSCs. Standard deviation wascalculated for population means for 3 donors. Mean % Stdev Lin-CD34⁺75.1 6.2 Lin-CD34⁻CD38⁻ 9.8 2.4 Lin-CD34⁻CD133⁺ 0.9 0.2 Lin-CD34⁻CD117⁺7.2 0.5

6.3. Example 3 Expansion of CD34⁺ Hematopoietic Cell Populations

The CD34⁺ cell content of human umbilical cord blood (UCB) units isoften not sufficient to provide for hematopoietic cell transplants inadult patients. Ex-vivo expansion of CD34⁺ cells from UCB is oneapproach to overcome this CD34⁺ cell dose limitation. This Exampledemonstrates expansion of CD34⁺ cells using a specific immunomodulatorydrug, 4-(Amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-1,3-dione(referred to in this Example as pomalidomide).

The ability of pomalidomide to enhance the expansion of human UCBderived CD34⁺ cells in a short-term serum-free, cytokine supplementedculture system was evaluated. CD34⁺ progenitor cells were enriched fromcryopreserved UCB units to >90% purity and seeded (104 CD34⁺ cells) in 1mL of growth medium, which consists of IMDM plus serum substitute BIT(BSA, recombinant human insulin and transferrin, 20%), in the presenceof SCF (50 ng/mL), Flt-3 ligand (50 ng/mL), and IL-3 (10 ng/mL).Pomalidomide, dissolved in DMSO, was supplemented at 2.7 μg/mL. Theculture was incubated at 37° C., 5% CO₂ for 12 days, with fresh mediumadded at day 7. Pomalidomide-free cultures with or without DMSO (0.05%v/v) were used as controls.

In one experiment, pomalidomide supplementation resulted insignificantly higher CD34⁺ expression in the expanded population withoutimpacting total nucleated cell expansion (200-350 fold). CD34⁺ phenotypein the pomalidomide-expanded population was 40-60%, compared with 10-30%in the control. Additionally, pomalidomide appeared to down-regulateCD38 expression on cultured cells. Pomalidomide-expanded CD34+ cellswere primarily CD38 negative (95%) and expressed lower levels of CD 133(15% vs. 40% in the control). Pomalidomide-expanded CD34+ cellsdemonstrated substantial improvement in cumulative colony forming unitsrelative to expanded controls. In another, similar, experiment,pomalidomide supplementation was confirmed to result in significantlyhigher CD34⁺ expression in the expanded population without impactingtotal nucleated cell expansion (200-350 fold). CD34⁺ phenotype in thepomalidomide-expanded population was 40-60%, compared with 10-30% in thecontrol (FIG. 3). Additionally, pomalidomide appeared to down-regulateCD38 expression on cultured cells. Pomalidomide-expanded CD34⁺ cellswere primarily CD38 negative (97%) and expressed lower levels of CD133(11.5% vs. 32.3% in the control). Pomalidomide-expanded CD34⁺ cellsdemonstrated substantial improvement in cumulative colony forming unitsrelative to expanded controls.

The pomalidomide-based CD34⁺ expansion process was scaled up todemonstrate the production of a larger number of CD34⁺ cells. CD34⁺cells were seeded in 10⁴/mL pomalidomide-supplemented medium in aflexible, gas-permeable fluorocarbon culture bag (American Fluoroseal).After 7 days of incubation, the culture was centrifuged and exchangedwith fresh pomalidomide-supplemented medium at three times the initialvolume. By day 12, TNC and CD34⁺ expansion were 350 (range: 250-700) and200 (range: 100-450) fold, respectively (FIG. 4). Viability was 86%(range: 80-90%) by trypan blue. A total of 20 million CD34⁺ cells wereharvested. These results demonstrate that pomalidomide significantlyenhanced the ex-vivo expansion of placental derived CD34⁺ progenitorsand that the process can produce a sufficient amount of CD34⁺ cells forerythrocytic differentiation.

6.4. Example 4 Feeder Cell-Free Expansion and Differentiation ofHematopoietic Stem Cells into Erythrocytes

This Example demonstrates continuous expansion and differentiation ofhematopoietic stem cells or precursor cells into erythrocytes usingmedium comprising SCF, IL-3 and Epo, and lacking Fms-like tyrosinekinase 3 ligand (FLT-3L), thrombopoietin (Tpo) and IL-11.

CD34⁺ cord blood cells were cultured in the following mediumformulations and aliquots of cells were taken for assessment of cellcount, cell viability and characterization of erythrocyticdifferentiation.

C medium: IMDM medium supplemented with 1% deionized BSA (Cat# A4919,Sigma), 120 μg/mL iron-saturated human transferrin (Cat# T0665, Sigma),900 ng/mL ferrous sulfate (Cat# F8048, Sigma), 90 ng/mL ferric nitrate(Cat# F8508, Sigma) and 10 μg/mL insulin (Cat# I0908, Sigma), 100 ng/mlSCF, 1 μM hydrocortisone (Cat# H0135, Sigma), 5 ng/mL IL-3 and 3 IU/mlEpo (Cat#287-TC, R&D Systems).

E1 medium: serum-free medium (STEMSPAN®, Cat#09650, Stem CellTechnologies, Vancouver, Canada) supplemented with 2 IU/mL Epo, 1 μMsynthetic glucocorticoid dexamethasone (Dex, Cat# D4902, Sigma, StLouis, Mo.), 40 ng/mL insulin-like growth factor 1 (IGF-1,Cat#291-G1-250, R&D Systems, Minneapolis, Minn.), 100 ng/mL SCF, and 40μg/mL lipids (cholesterol-rich lipid mix; Cat# C7305-1G, Sigma, StLouis, Mo.).

E2 medium: serum-free medium STEMSPAN® was supplemented with 2 IU/mLEpo, 1 μM hydrocortisone, 40 ng/mL IGF-1, 100 ng/mL SCF, and 40 μg/mLlipids.

E3 medium: serum-free medium STEMSPAN® was supplemented with 3 IU/mLEpo, 1 μM Dex, 40 ng/mL IGF-1, 100 ng/mL SCF, 5 ng/mL IL-3 and 40 μg/mLlipids.

E4 medium: serum-free medium STEMSPAN® was supplemented with 3 IU/mLEpo, 1 μM hydrocortisone, 40 ng/mL IGF-1, 100 ng/mL SCF, 5 ng/mL IL-3and 40 μg/mL lipids.

FIG. 5 shows the 21-day cell expansion in medium formulations C and E1.In summary, levels of cell expansion were up to 2.5×10⁵ fold with a meanof 7.0×10⁴ fold (n=13), levels of CD235A⁺ cells up to 37.6%, and levelsof enucleation up to 28.1% in C medium; levels of cell expansion were upto 2.6×10⁵ fold with a mean of 1.0×10⁵ fold (n=10), levels of CD235A⁺cells up to 92.9%, and levels of enucleation up to 48.2% in E medium.Cells expanded in these media all exhibited greater than 90% viability.Cells expanded in E1 medium exhibited the highest erythroiddifferentiation represented by the highest proportion of CD235A⁺ cellsand enucleated cells.

Cell expansion in 3 more medium formulations E2, E3 and E4 was examined(FIG. 6). When the cultures reached day 21 and further to day 28, cellsin E1 medium and E2 medium showed a growth plateau, while high cellproliferation was seen in E3 medium and E4 medium. Cells cultured for 21days were subjected to immunophenotypic characterization, FACS-basedanalyses of enucleation (TO-PRO-3, Cat# T3605, Invitrogen) andproduction of fetal hemoglobin (HbF-PE, Cat#560041, BD Biosciences) andadult hemoglobin (HbA-FITC, Cat# sc-21757, Santa Cruz)) (Table 3).

Table 3A, 3B. Characterization of day 21-cultured cells. (A)Immunophenotypic characterization; (B) Characterization of enucleation,HbF and HbA. Standard deviation was calculated for population means for3 donors.

TABLE 3A % CD34+ % CD38+ % CD117+ % CD133+ % CD71+ % CD36+ % CD235a MeanSTDEV Mean STDEV Mean STDEV Mean STDEV Mean STDEV Mean STDEV Mean STDEVE1 0.8 0.7 1.1 0.8 14.6 5.8 0.9 0.4 95.4 4.5 98.1 0.6 87.6 3.4 E2 0.40.2 0.7 0.1 14.4 6.3 0.8 0.2 95.5 1.7 97.9 0.4 85.6 3.1 E3 0.5 0.2 0.80.4 16.2 6.9 0.9 0.4 97.1 1.3 97.5 1.0 78.7 2.6 E4 0.2 0.1 0.5 0.1 17.78.2 0.7 0.3 96.5 1.4 96.3 1.0 70.8 2.9

TABLE 3B % Enucleation % HbF % HbA Mean STDEV Mean STDEV Mean STDEV E147.67 5.17 90.10 2.33 39.82 17.34 E2 42.93 4.63 90.07 1.15 34.33 16.61E3 41.97 6.12 91.90 0.60 37.36 16.42 E4 33.30 7.50 86.77 2.04 27.0714.10

Additionally, FACS analysis of HSCs markers (including CD34, CD117, andCD133) and erythocytic markers (including CD235A, CD36, and CD71) wasperformed using aliquots of cultures at serial time intervals ofcultures in E3 medium. A commitment to the erythroid lineage was evidentby day 7, as the expression of immature progenitor markers diminishedfor the CD34⁺ cell population, as wall as for the CD117⁺ and CD133⁺ cellpopulations, while 56% of the expanded cells were CD235A⁺. Thesubsequent terminal differentiation was demonstrated by continuouslyincreased expression of CD235A through days 7, 14, 21 and 28, increasedpresence of the enucleated cells, particularly between days 14 and 21,and increased production of both HbF and HbA through days 7, 14, 21 and28. At day 21 of cultures, nearly 50% of the expanded cells wereenucleated, 80% were HbF⁺, and 30% were HbA⁺.

Cells isolated by positive selection procedure and negative selectionprocedure were examined for expandability and differentiation in Bmedium (Table 4). Positively selected cells yielded average foldexpansion of 2.2×10⁴±2.0×10⁴; proportion of CD235⁺ cells was32.2%±14.8%; proportion of enucleated cells was 23.9%±7%. Negativelyselected cells yielded average fold expansion of 4.0×10⁴±3.8×10⁴;proportion of CD235⁺ cells was 40.9%±11.0%; proportion of enucleatedcells was 19.1%±4.8%. Based on the above results, no significantdifference of fold expansion and differentiation was observed for cellsderived from the positive and negative cell selection procedures.

TABLE 4 Evaluation of cells derived from positive and negative cellisolation procedures. Standard deviation was calculated for populationmeans for 3 donors. Positive Selection Negative Selection Average STDEVAverage STDEV Fold Expansion 2.20E+04 2.00E+04 4.00E+04 3.80E+04 %CD235+ 32.2 14.8 40.9 11 % Enucleation 23.9 7 19.1 4.8

6.5. Example 5 Effects of Cell Density on HSCs Expansion

HSCs were obtained from cord blood as described in Example 1. HSCcultivation was then initiated by culturing the enriched CR-derived HSCsE3 medium as described in Example 4. Every 3 to 4 days, the culturedcells were subjected to cell counting and characterizations. Thecultured cells were then diluted to desired densities using freshmedium.

Six cell densities were examined for effects on cell proliferationthroughout 26-day cultivation in E3 medium (FIG. 7 & Table 5). Cellsmaintained at a range of 2 to 5×10⁴ cells/mL showed the highestproliferation. Cells kept at >5×10⁵ cells/mL showed slower expansion.

TABLE 5 Effects of cell density on cell expansion Density D0 D9 D12 D14D16 D19 D21 D23 D26 1 2 × 10{circumflex over ( )}4 1.00E+00 4.22E+017.73E+02 1.09E+04 6.96E+04 9.27E+05 5.78E+06 4.35E+07 2.80E+08 2 5 ×10{circumflex over ( )}4 1.00E+00 2.92E+01 7.29E+02 5.81E+03 6.09E+047.51E+05 3.51E+06 9.74E+07 4.84E+08 3 1 × 10{circumflex over ( )}51.00E+00 3.51E+01 5.03E+02 3.33E+03 3.15E+04 4.84E+05 1.76E+06 5.14E+071.70E+08 4 2 × 10{circumflex over ( )}5 1.00E+00 2.79E+01 3.43E+021.82E+03 1.46E+04 2.14E+05 8.03E+05 1.76E+07 7.48E+07 5 5 ×10{circumflex over ( )}5 1.00E+00 3.19E+01 1.59E+02 4.35E+02 2.11E+031.71E+04 5.60E+04 6.49E+05 2.71E+06 6 1 × 10{circumflex over ( )}61.00E+00 2.97E+01 8.74E+01 1.70E+02 5.15E+02 2.54E+03 5.71E+03 4.14E+041.27E+05

6.6. Example 6 CD34⁺ Cells Derived from Placenta and Bone Marrow inExpansion and Differentiation into Erythrocytes

In this Example, a comparison of cell expansion and differentiationpotential between CB and bone marrow (BM) CD34⁺ cells in E3 medium wasperformed. Cells derived from 3 units of BM or CB as described inExample 1 were used in the evaluation studies. CB CD34⁺ cells showed ahigher proliferation potential compared with BM CD34⁺ cells (FIG. 8),while differentiation potential was comparable (Table 6A). Theproportions of cells containing adult hemoglobin (HbA) and fetalhemoglobin (HbF) (as compared to the total number of cells containinghemoglobin) are shown in Table 6B.

Table 6A, 6B. Comparison of differentiation potential of BM and CBderived CD34+ cells. Standard deviation was calculated for populationmeans for 3 donors.

TABLE 6A Day 21 fold expansion % CD235A+ BM CD34+ Average 8.09E+05 87.40STDEV 1.45E+05 3.08 CB CD34+ Average 1.01E+06 73.50 STDEV 8.06E+04 3.70

TABLE 6B HbA (%) HbF (%) BM CD34+ Average 73.83 57.38 STDEV 2.10 10.64CB CD34+ Average 22.99 88.70 STDEV 4.72 3.29

6.7. Example 7 Long Term Expansion and Differentiation into Erythrocytes

In this experiment, long term cell expansion was performed using E3medium (see Example 4, above) and CD34⁺ cells derived from a singledonor and mixed donors. Sustained cell growth up to 63 days with highRBC maturation efficiency was demonstrated (FIG. 9). At day 63, 1×10⁹-and 1.8×10⁹-fold expansion for the mixed donor and single donor cells,respectively, were observed, and 77% CD235A⁺ and 56% enucleation wereachieved for the single donor (Table 7).

TABLE 7 FACS analysis of CD235A⁺ cells and enucleation of the long termcultures derived from a single donor. Day 21 Day 28 Day 35 Day 42 Day 49Day 56 Day 63 % CD235A 65.3 78.6 74.8 63 55.4 61.4 70.4 % Enucleation39.3 41.3 40.3 47.3 46.5 53.9 51.9

FIG. 10 shows the ELISA analysis of HbF and HbA production in the longterm cultures derived from a single donor as compared to those inperipheral blood (PB) RBCs. In this experiment, 2×10⁵ cells collected atthe indicated time points were washed with PBS and spun at 2500×g for 10min. Cell pellets were lysed with 100□L M-PER Mammalian ProteinExtraction Reagent (Cat#78501, Thermo Scientific), and the mixture wasmixed gently for 10 minutes followed by centrifugation at 14,000×g for15 minutes. The supernatant was then transferred to a new tube forhemoglobin analysis using the ELISA kits: human hemoglobin ELISAquantitation set (Cat# E80-135) and human fetal hemoglobin ELISAquantitation set (Cat# E80-136). While 1 million PB erythrocytesproduced 245 ng HbA, cultured cells produced 1.3 μg HbA starting at day14 of cultivation.

Table 8 shows quantitative real-time PCR (qRT-PCR) analysis ofexpression of several genes in long time cultures derived from a singledonor. Aliquots of cells were taken at various time points and subjectedto qRT-PCR analysis using the 7900HT Fast Real-Time PCR System (AppliedBiosystems) and TaqMan® Gene Expression Assays of EKLF (AppliedBiosystems, Cat# Hs00610592_m1), GATA1 (Applied Biosystems, Cat#Hs00231112_m1), GATA2, HBβ (Applied Biosystems, Cat# Hs00758889_s1),HBγ_(Applied Biosystems, Cat# Hs00361131_g1), LMO2 (Applied Biosystems,Cat# Hs00277106_m1) and ZFPM1 (Applied Biosystems, Cat# Hs00419119_m1).The qRT-PCR analysis results showed the sustained growth anddifferentiation into erythrocytes were correlated with increasedexpression of HBβ, HBγ, and several transcription factors that arecrucial for erythrocytic differentiation, including EKLF, GATA1, LMO2and ZFPM1.

TABLE 8 qRT-PCR analysis of long term cultures derived from a singledonor. (A) Fold change of gene expression; (B) Gene description.Standard deviation was calculated for means of fold change for 2replicates. Table 8A. EKLF GATA1 GATA2 HBβ HBγ LMO2 ZFPM1 Mean STDEVMean STDEV Mean STDEV Mean STDEV Mean STDEV Mean STDEV Mean STDEV Day 01.0 0.2 1.0 0.1 1.0 0.1 1.0 0.3 1.0 0.1 1.0 0.3 1.0 0.2 Day 7 17.6 0.22.4 0.0 0.3 0.0 4.8 0.1 1.7 0.0 1.0 0.1 1.0 0.1 Day 14 45.5 0.4 6.2 0.71.0 0.0 23.9 1.4 19.8 0.1 2.7 0.2 2.4 0.1 Day 21 51.5 3.1 6.4 0.1 0.90.1 41.5 3.3 21.6 1.0 2.3 0.3 2.2 0.0 Day 28 64.5 14.1 6.9 0.2 1.2 0.046.2 1.2 26.0 1.8 3.6 0.2 3.3 0.6 Day 35 38.5 1.6 10.1 1.0 1.5 0.2 16.20.4 16.0 0.4 2.9 0.2 6.0 1.6 Day 42 31.4 1.9 5.9 0.0 0.8 0.0 8.2 0.112.2 0.2 1.7 0.0 1.5 0.2 Day 49 38.3 2.5 5.1 0.1 0.7 0.1 4.5 1.0 11.40.2 1.3 0.1 1.7 0.4 Day 56 46.0 2.5 5.8 0.1 0.6 0.0 6.2 0.5 25.0 1.8 1.50.1 2.4 0.1 Day 63 67.4 2.1 9.2 0.2 1.2 0.0 12.1 0.8 32.0 1.4 3.7 0.22.8 0.0 Table 8B. Symbol Description EKLF Kruppel-like factor 1(erythroid) GATA1 GATA binding protein 1 (globin transcription factor 1)GATA2 GATA binding protein 2 HBβ Hemoglobin, beta Hbγ Hemoglobin, gammaLMO2 LIM domain only 2 (rhombotin-like 1) ZFPM1 zinc finger protein,multitype 1

6.8. Example 8 Optimization of E3 Medium

In this Example, E3 medium was further optimized to improve HSCexpansion and differentiation into erythrocytes using a 3-level (3factors: SCF, Epo and IL-3) full factorial experiment design (FIG. 11).CD34⁺ cells were obtained as described in FIG. 1, and E3 medium wassupplemented with SCF, IL-3 and EPO shown in Table 9. Cell count,viability, proportion of CD235A⁺ cells and proportion of enucleatedcells were examined at the time points indicated in Table 10. Oneformulation (#7, 65 ng/mL SCF, 7 ng/mL IL-3 and 3 IU/mL Epo) showed anincreased proportion of CD235A⁺ cells and increased enucleation ascompared to the E3 medium formulation.

TABLE 9 Design of Experiment for E3 medium optimization ExperimentalDesign Condition# SCF (ng/mL) IL-3 (ng/mL) EPO (IU/mL) 1 65 7 1 2 65 3 13 135 3 1 4 135 7 1 5 100 5 2 6 65 3 3 7 65 7 3 8 135 3 3 9 135 7 3 E3100 5 3

TABLE 10 Summary of E3 medium optimization at Day 21. Means STDEVCondition# fold expansion % CD235A % Enucleation fold expansion % CD235A% Enucleation 1 6.5E+05 74.7 13.2 4.1E+04 21.2 7.0 2 6.1E+05 71.2 15.21.1E+05 0.1 0.1 3 6.3E+05 69.0 18.7 4.9E+04 5.1 4.6 4 6.8E+05 67.3 19.35.3E+03 1.4 1.2 5 6.0E+05 65.6 17.9 1.0E+04 3.6 1.6 6 6.0E+05 76.9 18.64.9E+04 0.4 0.2 7 5.7E+05 77.3 24.8 1.0E+05 0.0 1.5 8 6.3E+05 76.0 17.29.9E+04 0.1 0.9 9 5.6E+05 64.2 14.6 1.4E+04 3.6 5.9 E3 6.8E+05 63.5 18.27.1E+03 0.0 0.0

This study has identified the differential effects of SCF and Epointeractions on HSC expansion and differentiation into erythrocytesduring a 21-day cell expansion experiment (FIGS. 12A-12C). Epo workssynergistically to increase early expansion at a high level of SCF, butreduces expansion at low levels of SCF. At low SCF levels, Epo is moreefficient at increasing CD235A expression; while at high SCF levels, Eporeduces erythrocytic differentiation.

In a second DOE study, a 3-level (SCF, Epo and cell density) fullfactorial experiment design was utilized to assess the interactions ofcell seeding density to SCF and/or Epo (FIG. 13A). CD34⁺ cells wereobtained as described in FIG. 1, and E3 medium was supplemented with SCFand EPO with different cell densities shown in Table 11. It wasdemonstrated that significantly enhanced expansion (nearly 10-fold) canbe achieved with lower Epo conditions (up to 3-fold), low cell density,and high SCF concentration (FIG. 13B). In addition, it was alsodemonstrated that erythrocytic differentiation can be improved using ahigher cell seeding density and lower SCF concentration (FIG. 13C).

TABLE 11 Design of Experiment for E3 medium optimization RunOrder SeedDensity (#cell/mL) EPO (IU/mL) SCF (ng/mL) 1 500000 5 1 2 50000 1 100 350000 5 100 4 50000 1 100 5 275000 3 50.5 6 275000 3 50.5 7 50000 5 1 8500000 5 100 9 50000 1 1 10 500000 1 1 11 500000 1 100 12 500000 5 1 13500000 1 100 14 50000 1 1 15 500000 1 100 16 50000 5 100 17 500000 1 118 50000 1 1 19 275000 3 50.5 20 500000 5 100 21 500000 5 100 22 50000 51 23 50000 5 1 24 50000 1 100 25 50000 5 100 26 500000 5 1 27 500000 1 1

6.9. Example 9 Method and Bioreactor for Generating Units ofErythrocytes

This Example provides a method of producing erythrocytes, and abioreactor that enables the production of units of mature erythrocytes.In this particular example, the bioreactor enables the production ofadministrable units of erythrocytes using a five-step process. In thefirst step, hematopoietic cells, e.g., CD34⁺ cells, are isolated. In thesecond step, the CD34⁺ cells are expanded using an immunomodulatorycompound, e.g., pomalidomide. In the third step, the CD34⁺ cells areexpanded in the bioreactor exemplified herein, in a co-culture withadherent placental stem cells, in conjunction with removal oflineage-committed cells. Fourth, remaining uncommitted hematopoieticcells are differentiated to erythrocytes. Finally, in the fifth step,erythrocytes are isolated and collected into administrable units.

Steps 1 and 2, the isolation and initial expansion of hematopoieticcells, are accomplished as described in Examples 3 and 4, above.

Steps 3 and 4 are accomplished using a bioreactor. The bioreactorcomprises a hollow fiber chamber (1) seeded with placental stem cells(2) and an element for gas provision to the medium (3). The bioreactorfurther comprises a coupled cell sorter/separator element (4) thatallows for the continuous separation of committed hematopoietic cells,fully-differentiated erythrocytes, or both. The cell separation elementcan separate the cells from the hematopoietic cells using, e.g.,magnetic cell separation or fluorescence-activated cell separationtechniques.

To initiate cell culture, approximately 5×10⁷ hematopoietic cells, e.g.,CD34⁺ hematopoietic cells, are inoculated into the bioreactor.

6.10. Example 10 Collection of Erythrocytes

This Example exemplifies several methods of the separation oferythrocytes from other lineage committed cells.

Method 1: Erythrocytes, e.g., erythrocytes collected from the cellseparation element of the bioreactor exemplified herein, and hetastarchsolution are mixed 3:1 (v:v) in a Baxter collection bag and placed in anupright position on a plasma extractor. Erythrocytes sediment after 50to 70 minutes. Non-sedimented cells are forced out by the plasmaextractor. Sedimented erythrocytes left in the bag can be furthercollected by centrifugation at 400×g for 10 minutes. After removing thesupernatant, erythrocytes are resuspended in an appropriate amount ofdesired medium.

Method 2—Immunomagnetic separation: Glycophorin A⁺ cells, e.g.,erythrocytes collected from the cell separation element of thebioreactor exemplified herein, are magnetically labeled with GlycophorinA (CD235a) MicroBeads (Miltenyi Biotech). The cell suspension is thenloaded into a tube which is placed in the magnetic field of an EASYSEP®magnet. The magnetically labeled Glycophorin A⁺ cells are retainedinside the tube, while the unlabeled cells are poured off the tube.After removal of the tube from the magnetic field, the magneticallyretained Glycophorin A⁺ cells can be separated from the magnetic beadsand resuspended in an appropriate amount of desired medium.

Method 3—Flow cytometry cell separation: Erythrocytes, e.g.,erythrocytes collected from the cell separation element of thebioreactor exemplified herein, in 500 μL PBS/FBS with 1 μL Fc Block(1/500). 150 μL of the cell suspension is added to each well of a 96well V-bottom dish. 50 μL 1° Ab Master Mix (the mix is a 1/25 dilutionof each primary Ab in PBS/FBS) is added to the cells. One well isincluded with a combination of isotype controls for setting voltage, aswell as one well for each of the primary Ab as single positive controlsfor setting compensation. The cells are incubated 60 min at 4° C., thencentrifuged at 1500 RPM for two minutes. The supernatant is discarded.The wells are washed with 200 μL PBS/FBS to each well, and mixed bypipetting up and down. The cells are then immediately spun at 1500 RPM×2min; the supernatant is discarded. 150 μL of secondary Ab (i.e.Streptavidin-TC) Master Mix is added, and incubated 30 min at 4° C.,followed by centrifugation at 1500 RPM for 5 minutes. The pellet isresuspended in 200-500 μL of PBS/FBS and transferred to 5 mL flow tubes.Cells are then separated using a flow cytometer.

Method 4: Medium comprising erythrocytes, in continuous flow between thecell culture element and cell separation element, is deoxygenated byreducing or turning off the supply of oxygen from the gas provisionelement, and turning on a magnet in the cell separation element. Mediumis passed through the cell separation element for a sufficient time forthe magnetic field of the magnet to collect erythrocytes to a surface inthe cell separation element. Once a predetermined number of erythrocytesare collected, or collection has proceeded for a predetermined amount oftime, the medium is reoxygenated, releasing the erythrocytes from thesurface.

Method 5: Sedimentation based erythrocyte enrichment. Erythrocyteenrichment can be performed by centrifugation at 3000 rpm for 15 minwith break off. Leukocytes (top white layer), immature erythrocytes(middle pink layer), and erythrocytes (bottom red layer) can beseparated. Erythrocytes are then collected from the bottom andresuspended in an appropriate amount of desired medium.

6.11. Collection of Erythrocytes

This Example demonstrates the collection of erythrocytes using flowcytometry.

Erythrocyte enrichment was performed by FACSAria sorting to selecterythrocytes by cell size using light scatter (forward and side scatter,FIG. 14 and Table 12) or enucleation using DRAQ5 labeling (linear APCchannel fluorescence, FIG. 15 and Table 13).

After cell sorting by FACSAria, cells gated by P1, P2 and P3 wereassessed for proportion of HbA⁺, enucleation and CD235A⁺ by flowcytometry. P1 cells showing smallest cell size exhibited highest percentHbA⁺, percent enucleation and percent CD235A⁺, and therefore were mostlyerythrocytes.

TABLE 12 Characterization of sorted erythrocyte populations by cell sizeLiving Samples cells P1 P2 P3 % HbA+ % Enucleation % CD235A+ Presort63.50% 26.18% 34.34% 13.39% 28.2 22.2 69.8 Sorted P1 50.34% 62.04% — —30.3 61.5 92.1 P2 49.66% — 53.31% — 14.31 33.7 76.4 P3 54.01% — — 30.24%18.12 2.64 74.84

In Table 12, cells were stained by cell permeable DNA-interactive agentDRAQ5 (Cell Signaling, Catalog No. #4084) followed by cell sorting usingFACSAria, cells gated by Q1 and Q2 were assessed for proportion of HbA⁺,enucleation and CD235A⁺ by flow cytometry. Q1 cells that were negativefor DRAQ staining, exhibited higher percent HbA⁺, percent enucleationand percent CD235A+ compared with Q2 cells, and therefore were mostlyerythrocytes.

TABLE 13 Characterization of sorted erythrocyte populations by DRAQ5staining Living cells APC+ APC− % HbA+ % Enucleation % CD235A+ Presort64.51% 13.76% 47.83% 28.2 22.2 69.8 Sorted Q1 DRAQ5 49.69% 82.12% 24.9360.7 91.8 Negative Q2 DRAQ5 30.20% 39.72% 18.96 15.4 65.7 Positive

As depicted in Table 13, cells were stained by cell permeableDNA-interactive agent DRAQ5. After cell sorting by FACSAria, cells gatedby QP1, P2 and QP3 were assessed for proportion of HbA⁺, enucleation andCD235A⁺ by flow cytometry. QP1 cells that were negative for DRAQstaining, showing smallest cell size exhibited the highest percent HbA⁺,percent enucleation and percent CD235A⁺ compared with Q2 cells, andtherefore were predominantly erythrocytes.

6.12. Example 11 Bioreactor for Producing Erythrocytes

This Example describes a bioreactor design that allows for improvedproduction of erythrocytes from hematopoietic cells. The bioreactorcomprises a culturing element that comprises hollow fibers in whichhematopoietic cells are cultured. Hematopoietic cells, e.g.,hematopoietic progenitor cells, are supplied in a bag at 5×10⁵cells/dose, where one dose yields one unit of blood. The cells areexpanded in the presence of IMDM medium containing 50 ng/mL SCF, 3units/mL Epo, and 50 ng/mL IGF-1 added through a first port. Gasprovision (5% CO2 in air) occurs through a second port. The medium inwhich the hematopoietic cells are cultured is supplemented withpomalidomide at 2.7 μg/mL. During culturing, gas, medium metabolites andmedium pH in the culturing element is monitored continuously, and arereplenished or exchanged using a programmable control device asnecessary. pH of the medium in the culturing element is maintained atapproximately 7 and the culture temperature is maintained at about 37°C. Lineage-committed cells (i.e., differentiated cells) are continuouslyseparated and recovered from the culture medium using a cell separationelement. The bioreactor is equipped with an independent power supply toenable operation at a remote site, e.g., a site separate from a site atwhich hematopoietic cells are initially obtained.

The present disclosure, including devices, compositions and methods, isnot to be limited in scope by the specific embodiments described herein.Indeed, various modifications in addition to those described herein willbecome apparent to those skilled in the art from the foregoingdescription. Such modifications are intended to fall within the scope ofthe appended claims.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentdisclosure is not entitled to antedate such publication by virtue ofprior invention.

What is claimed is:
 1. A method of producing erythrocytes, comprisingexpanding a population of human hematopoietic cells in a medium in theabsence of feeder cells and in contact with one or more factors, whereina plurality of human hematopoietic cells within said population of humanhematopoietic cells differentiate into erythrocytes during saidexpanding; and isolating said erythrocytes from said medium, whereinsaid factors comprise SCF at a concentration of about 10 to about 10,000ng/mL, IL-3 at a concentration of about 0.01 to about 500 ng/mL, and Epoat a concentration of about 0.1 to about 10 IU/mL, wherein said mediumcomprises insulin-like growth factor 1 (IGF-1) at a concentration ofabout 1 to about 1000 ng/mL, wherein said medium does not comprise oneor more of Flt-3L, IL-11, thrombopoietin (Tpo), homeobox-B4 (HoxB4), ormethylcellulose, and wherein said SCF, IL-3 and Epo are not comprisedwithin an undefined component of said medium.
 2. The method of claim 1,wherein said SCF is present at a concentration of about 100 ng/mL. 3.The method of claim 1, wherein said IL-3 is present at a concentrationof about 5 ng/mL.
 4. The method of claim 1, wherein said EPO is presentat a concentration of about 2 to about 3 IU/mL.
 5. The method of claim1, wherein said hematopoietic cells are CD34⁺.
 6. The method of claim 1,wherein said medium further comprises lipids at a concentration of about1 to about 1000 μg/mL, wherein said lipids comprise a mixture of proteinand cholesterol; and wherein said medium comprises hydrocortisone at aconcentration of about 0.01 to about 100 μM, or dexamethasone at aconcentration of about 0.01 μM to about 100 μM.
 7. The method of claim6, wherein said IGF-1 is present at a concentration of about 20 to about100 ng/mL.
 8. The method of claim 6, wherein said lipids are present ata concentration of about 20 to about 100 μg/mL.
 9. The method of claim6, wherein said dexamethasone is present at a concentration of about 0.1to about 10 μM.
 10. The method of claim 6 wherein said medium comprisesabout 100 ng/mL SCF, about 3 IU/mL Epo, about 40 ng/mL IGF-1, about 1 μMDexamethasone, and 40 μg/ml lipids.
 11. The method of claim 6 whereinsaid medium comprises about 100 ng/mL SCF, about 2 IU/mL Epo, about 40ng/mL IGF-1, about 1 μM hydrocortisone, and 50 μg/mL lipids, whereinsaid lipids comprise a mixture of protein and cholesterol.
 12. A methodof producing erythrocytes, comprising expanding a population of humanhematopoietic cells in a medium in the absence of feeder cells, whereina plurality of human hematopoietic cells within said population of humanhematopoietic cells differentiate into erythrocytes during saidexpanding; and isolating said erythrocytes from said medium, whereinsaid medium comprises about 100 ng/mL SCF, about 3 IU/mL Epo, about 40ng/mL IGF-1, about 1 μM dexamethasone, about 40 μg/ml lipids, and about5 ng/mL IL-3, wherein said medium does not comprise one or more ofFlt-3L, IL-11, thrombopoietin (Tpo), homeobox-B4 (HoxB4), ormethylcellulose, and wherein said SCF, IL-3 and Epo are not comprisedwithin an undefined component of said medium.
 13. A method of producingerythrocytes, comprising expanding a population of human hematopoieticcells in a medium in the absence of feeder cells, wherein a plurality ofhuman hematopoietic cells within said population of human hematopoieticcells differentiate into erythrocytes during said expanding; andisolating said erythrocytes from said medium, wherein said mediumcomprises about 100 ng/mL SCF, about 3 IU/mL Epo, about 40 ng/mL IGF-1,about 1 μM hydrocortisone, about 40 μg/mL lipids, and about 5 ng/mLIL-3, wherein said medium does not comprise one or more of Flt-3L,IL-11, thrombopoietin (Tpo), homeobox-B4 (HoxB4), or methylcellulose,and wherein said SCF, IL-3 and Epo are not comprised within an undefinedcomponent of said medium.
 14. A method of producing erythrocytes,comprising expanding a population of human hematopoietic cells in amedium in the absence of feeder cells, wherein a plurality of humanhematopoietic cells within said population of human hematopoietic cellsdifferentiate into erythrocytes during said expanding; and isolatingsaid erythrocytes from said medium, wherein said medium comprises about10 to about 10,000 ng/mL SCF, about 0.01 to about 500 ng/mL IL-3, about0.1 to about 10 IU/mL Epo, about 40 ng/mL IGF-1, about 1 μMdexamethasone, and about 40 μg/mL lipids, wherein said medium does notcomprise one or more of Flt-3L, IL-11, thrombopoietin (Tpo), homeobox-B4(HoxB4), or methylcellulose, and wherein said SCF, IL-3 and Epo are notcomprised within an undefined component of said medium.